JP2016504417A - Intein-mediated protein purification - Google Patents

Abstract

The present invention comprises contacting a first fusion protein containing a desired protein (POI) fused to the C-terminus of an intein C fragment with a second fusion protein containing an intein N fragment and a purification tag, Forming a complex of the fusion protein of and a second fusion protein, cleaving the POI from the intein C fragment, releasing the protein from the complex, and isolating the POI Including methods, compositions, uses and kits for purifying POI. The present invention also includes fusion proteins and vectors.

Description

  The present invention relates generally to the fields of protein purification and protein cleavage.

STATEMENT OF FEDERALLY SPONSORED RESEARCH This invention was made with US government support by FA95550-12-1-0330 provided by the US Air Force / Air Force Science Laboratory and 1150478 provided by the National Science Foundation. The government has certain rights in the invention.

  Without limiting the scope of the invention, the background of the invention relating to intein-mediated protein purification is described.

  US 2006/0141570 (filed Nov. 16, 2005) is a fusion protein comprising a product protein domain, an intein and at least one aggregate protein domain, wherein the aggregate protein domain comprises: Disclosed is the purification of recombinant proteins, including proteins that can be specifically involved in granules of polyhydroxyalkanoate (PHA) and carried out by expression in host cells.

  European Patent Application No. 1117693B1 (filed Sep. 30, 1999) selectively cleaves a target protein fused to an intein with a thiol reagent in a first step to form a carboxy-terminal thioester of the target protein; An in vitro method for producing a semisynthetic fusion protein that releases a target protein from an intein is disclosed. In subsequent steps, the desired synthetic protein or peptide having a cysteine at the amino terminus is linked to the target protein. Standard thiol reagents such as DTT or thiol reagents optimized for ligation such as odorless MESNA can be used in the first step. This method is said to link the desired peptide directly to a thioester bond that binds the target protein to the intein. An in vivo modification of this method is said to allow the production of cytotoxic proteins, introducing an inactive truncated protein fused to intelin in vivo, and then selectively selecting the fusion product. To cleave and restore the original activity of the cytotoxic protein, the synthetic protein or peptide is then linked to the carboxy terminal thioester of the target protein.

  European Patent Application No. 1151117A4 (filed Aug. 10, 2005) discloses a method for linking expressed proteins utilizing an intein, for example the RIR1 intein of Methanobacterium thermotrophicum. Yes. Mth RIR1 intein constructs in which either the intein C-terminal asparagine or the N-terminal cysteine is replaced by alanine are used for the fusion of two or more expressed proteins of proteins with a specified N-terminus, eg, cysteine. Isolation can be facilitated. The method comprises generating a second target protein having a designated N-terminus via an intein such as a target protein tagged with a C-terminal thioester and a modified Mth RIR1 intein; and linking these proteins including. Similar methods are provided for producing cyclic or polymerized proteins. Also provided are modified inteins engineered to cleave each C-terminus or N-terminus, and DNAs and plasmids encoding these modified inteins.

US Patent Application No. 2006/0141570 European Patent Application No. 1117693B1 European Patent Application No. 1151117A4

  In the present invention, a first fusion protein containing a desired protein (POI; protein of interest) fused to the C-terminus of an intein C fragment is contacted with a second fusion protein containing an intein N fragment and a purification tag. Forming a complex of the first fusion protein and the second fusion protein, cleaving the POI from the intein C fragment, releasing the protein from the complex, and And a step of purifying the POI. In a particular embodiment, the intein is split intein, a naturally split intein DnaE of Nostoc punctiforme and / or Ssp of Synechocystis sp., Aphanothece halophytica Aha, Aov of Aphanizomenon ovalisporum, Asp of Anabaena spp., Ava of Anabaena variabilis, Cylindrospermopsis raCiboroti (Cylindrospermopsis raCiboroti) Species Csp (CCYOllO), Cyanothece Species Csp (PCC8801), Crocosphaera watsonii Cwa, Microcystis et Maer (NIES843) of Microcystis aeruginosa, Mcht (PCC7420) -2 of Microcoleus chthonoplastes, Oli of Oscillatoria limnetica (long) of Synechococcus elongatas (PC) Synspococcus species Ssp (PCC7002), Thermocinecococcus elongatas Tel, Trichodesmium erythraeum Ter-3 and Thermocinecocus vulcanus coccus ulcanus cc Selected from the group consisting of Tvu. In certain embodiments, the intein C fragment is a mutation that significantly delays N-terminal cleavage, suppresses trans-splicing ability, and increases C-terminal cleavage rate and efficiency compared to a non-mutated intein C fragment. The C-intein fragment has an Asp118Gly mutation within the C-intein fragment, and / or the intein C fragment comprises the amino acid sequence of SEQ ID NO: 37. In certain aspects, the purification tag is located at the intein split junction at the C-terminus of the intein N fragment, the intein N fragment has a mutation that eliminates N-terminal cleavage activity, and / or the intein N fragment is a sequence It contains the amino acid sequence of number 39. In a particular embodiment, the purification tag is an affinity tag selected from the group consisting of a chitin binding domain (CBD), 6x histidine, maltose binding domain (MBP), glutathione-S-transferase (GST) and combinations thereof. is there. In certain embodiments, the purification tag is an affinity tag selected from the group consisting of SEQ ID NO: 38, and / or the second fusion protein is from the group consisting of SEQ ID NOs: 4, 10, 24, and combinations thereof. Contains a selected amino acid sequence. In certain embodiments, the purification tag is an elastin-like peptide (ELP) and / or the purification tag is a precipitation tag comprising the amino acid sequence of SEQ ID NO: 38. In certain aspects, the purification tag is a sedimentation tag, and the method further includes precipitating the complex, washing the complex, solubilizing the complex, and introducing intein cleavage. In certain embodiments, the precipitation of the complex and the washing of the complex are performed in the presence of one or more cleavage inhibitors. In certain embodiments, the purification tag is an affinity tag, and the method further includes binding the complex to an affinity resin capable of binding to the affinity tag, and washing the complex before the cleavage step. Washing with a liquid and inducing intein cleavage. In certain embodiments, complex binding and complex washing are performed in the presence of one or more cleavage inhibitors. In certain aspects, induction of intein cleavage is performed by a reducing agent or a chelating agent. In a particular embodiment, the induction of intein cleavage is a group consisting of a complex and ethyleneglycolaminoethylester tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), dipicolinic acid (DPA), nitrilotriacetic acid (NTA). Contacting with one or more chelating agents selected from: In certain aspects, the method further comprises incubating the complex with a first wash buffer prior to inducing cleavage, wherein the wash buffer inhibits cleavage and / or Zn 2+, Cu 2, Mg 2+. Including a cleavage inhibitor selected from the group consisting of Co2 +, Mn2 + and Fe2 +; and / or washing the complex with a first wash buffer prior to inducing cleavage comprising the wash buffer Comprising a cleavage inhibitor that inhibits the C-terminal cleavage reaction. In certain aspects, C-terminal protein cleavage is sulfur-induced C-terminal cleavage induction, DTT, Zn2 + chelator, trialkylphosphine (tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethanol, cysteine and the like Induction of C-terminal protein cleavage, including induction of sulfur-induced C-terminal cleavage in the presence of a cleavage inducer selected from the group consisting of combinations of the above, induction of intein cleavage by chelation of a cleavage inhibitor using a chelating agent In certain embodiments, when the purification tag is an affinity tag, the method further comprises binding the complex to an affinity resin, wherein the separation of the POI from the complex comprises Separating the POI from the complex-bound affinity resin and / or the purification tag is In this case, the method further comprises the step of sedimenting the complex, comprising forming a sedimented complex, and the separation of the POI from the complex comprises solubilization of the sedimented complex, whereby the solubilized complex is formed. And separating the POI from the solubilized complex, In certain embodiments, the method further comprises isolating the second fusion protein by dissociating the intein C fragment from the second fusion protein. In certain embodiments, the POI is a bioactive peptide, enzyme, enzyme inhibitor, enzyme catalytic site, DNA binding protein, isolated protein domain, receptor ligand, receptor, growth factor, cytokine, Consecutive or structural proteins, antibodies, antibody fragments, epitopes, epitope binding regions, antigens, allergens and desired protein sequences In certain embodiments, the purification tag is an affinity tag, and the method further includes binding the complex to an affinity resin prior to inducing C-terminal protein cleavage, and a second fusion protein. Regenerating the affinity resin by dissociating the intein C fragment from the method In certain embodiments, the method further regenerates the second fusion protein by dissociating the intein C fragment from the second fusion protein. And re-contacting the regenerated second fusion protein with the first fusion protein In certain embodiments, when the purification tag is an affinity tag, the second fusion protein is chitin beads, Binding to an affinity resin selected from the group consisting of nickel resin, amylose resin, glutathione and combinations thereof, If the produced tag is a precipitation tag that mediates precipitation of the second fusion protein, the complex is precipitated.

  The present invention provides a first fusion protein comprising a desired protein (POI) and an intein C fragment, wherein the POI is fused to the C-terminus of the intein C fragment and the intein is naturally split in DnaE. The intein C fragment has an Asp118Gly mutation in the intein C fragment and providing a second fusion protein comprising the intein N fragment and a purification tag, wherein the purification tag is an intein at the C-terminus of the intein N fragment. Inserting the split junction and having the mutation that causes the intein N fragment to lose N-terminal cleavage activity, and contacting the first fusion protein and the second fusion protein in a binding buffer, wherein The two fusion proteins bind to the resin that binds to the purification tag, and the purification tag C-terminal protein of the first fusion protein that can bind specifically to the resin, forms a complex of the first fusion protein and the second fusion protein, and the binding buffer is between the POI and the intein C fragment Inhibiting the cleavage, inducing C-terminal protein cleavage of the first fusion protein between the POI and the intein C fragment, thereby releasing the POI, and C of the first fusion protein and the intein C fragment. Separating the POI from the terminus, and an embodiment of a method for purifying the POI.

  The present invention also provides a first fusion protein comprising a desired protein (POI) and an intein C fragment, wherein the POI is fused to the C-terminus of the intein C fragment, and the intein DnaE is naturally split. The intein C fragment has an Asp118Gly mutation in the intein C fragment and a second fusion protein comprising an intein N fragment and a precipitation tag, wherein the precipitation tag is attached to the C-terminus of the intein N fragment. Inserting an intein split junction with a mutation that causes the intein N fragment to lose N-terminal cleavage activity, and contacting the first and second fusion proteins in a binding buffer. A complex of the first fusion protein and the second fusion protein is formed Binding buffer inhibits C-terminal protein cleavage of the first fusion protein between POI and intein C fragment, precipitating the complex of the first fusion protein and the second fusion protein, Solubilizing the body in a low salt buffer, inducing C-terminal proteolytic cleavage of the first fusion protein between the POI and the intein C fragment, thereby releasing the POI, and the first fusion protein And separating the POI from the complex of the second fusion protein by a second round of sedimentation.

  The present invention includes the desired protein (POI) and the intein C fragment, the POI is fused to the C-terminus of the intein C fragment, the intein is a naturally split intein DnaE, and the intein C fragment is within the intein C fragment. Embodiments of fusion proteins having mutations are included. In a particular embodiment, the fusion protein comprises SEQ ID NO: 37. In certain embodiments, the POI is a bioactive peptide, enzyme, enzyme inhibitor, enzyme catalytic site, DNA binding protein, isolated protein domain, receptor ligand, receptor, growth factor, cytokine, antibody, antibody fragment, epitope Selected from contiguous or overlapping fragments of the epitope binding region, antigen, allergen and the desired protein sequence.

  The present invention implements a fusion protein comprising an intein N fragment and a purification tag, wherein the purification tag is located at the intein split junction at the C-terminus of the intein N fragment and the intein N fragment has a mutation that eliminates N-terminal cleavage activity Includes form. In particular aspects, the fusion protein comprises SEQ ID NO: 10, 24, SEQ ID NO: 21, 22, SEQ ID NO: 4, SEQ ID NO: 23, or combinations thereof.

  The present invention relates to a vector comprising a first DNA element encoding the C-terminus of an intein C fragment operably linked to a promoter, wherein the intein C fragment is not mutated compared to an intein C fragment. Vector having a cloning site that has a mutation that suppresses C and increases C-terminal truncation, and allows the vector to insert the intein C fragment of the second DNA element encoding the desired protein (POI) Embodiments are included. In a particular embodiment, the intein is a naturally split intein DnaE of Nostock punctiforme and the C-intein fragment has an Asp118Gly mutation within the C-intein fragment. In certain embodiments, the first DNA element encodes the amino acid sequence of SEQ ID NO: 37 and / or the first DNA element comprises SEQ ID NO: 40. In certain embodiments, the POI is a bioactive peptide, enzyme, enzyme inhibitor, enzyme catalytic site, DNA binding protein, isolated protein domain, receptor ligand, receptor, growth factor, cytokine, antibody, antibody fragment, epitope Selected from contiguous or overlapping fragments of the epitope binding region, antigen, allergen and the desired protein sequence.

  The present invention is a vector comprising a DNA element encoding a fusion protein comprising an intein N fragment operably linked to a promoter and a purification tag, wherein the purification tag is located at the intein split junction at the C-terminus of the intein N fragment And an embodiment of the vector in which the intein N fragment has a mutation that abolishes N-terminal cleavage activity. In certain aspects, the purification tag is an affinity tag and / or the purification tag is a precipitation tag. In certain embodiments, the DNA element comprises SEQ ID NO: 23 or SEQ ID NO: 41.

  The present invention relates to a first vector comprising a first DNA element encoding the C-terminus of an intein C fragment operably linked to a promoter, as compared to an intein C fragment in which the intein C fragment is not mutated. Has mutations that suppress N-terminal truncation and increase C-terminal truncation, allowing the first vector to insert the intein C fragment of the second DNA element encoding the desired protein (POI) into the C-terminus A second vector comprising a first vector having a cloning site that comprises a second DNA element encoding a fusion protein comprising an intein N fragment operably linked to a promoter and a purification tag, wherein the purification tag is an intein Located at the intein split junction at the C-terminus of the N fragment, the intein N fragment loses N-terminal cleavage activity A fusion protein comprising an intein N fragment and a purification tag located at the intein split junction at the C-terminus of the intein N fragment, wherein the intein N fragment abolishes N-terminal cleavage activity A method for isolating a POI comprising a fusion protein having a mutation, instructions for inserting a DNA element encoding the POI into the cloning site of the first vector, and instructions for isolating the POI. Includes kit embodiments.

  The present invention provides a first fusion protein comprising contacting a first fusion protein containing a desired protein (POI) fused to the C-terminus of an intein C fragment with a second fusion protein containing an intein N fragment and a purification tag. And a second fusion protein complex, wherein the intein C fragment is significantly delayed in N-terminal cleavage compared to an intein C fragment in which the intein C fragment is not mutated, the trans-splicing ability is suppressed, and the C-terminal cleavage rate is reduced. Carrying out a method of purifying POI, comprising: cleaving POI from an intein C fragment, and releasing the protein from the complex; and isolating the POI. Includes form. In a particular embodiment, the intein is a naturally split intein DnaE and the C-intein fragment has an Asp118Gly mutation within the C-intein fragment.

  The present invention provides a first fusion protein comprising a desired protein (POI) and an intein C fragment, wherein the POI is fused to the C-terminus of the intein C fragment and the intein is a naturally split intein DnaE. Yes, the intein C fragment has an Asp118Gly mutation in the intein C fragment, and provides a second fusion protein comprising the intein N fragment and a purification tag, wherein the intein N fragment abolishes N-terminal cleavage activity Contacting the first fusion protein and the second fusion protein in a binding buffer, wherein the second fusion protein binds to a resin that binds to the purification tag, and the purification tag is A first fusion protein and a second fusion tongue that can specifically bind to a purified resin A protein complex is formed and the binding buffer inhibits C-terminal protein cleavage of the first fusion protein between POI and intein C fragment; and first fusion protein between POI and intein C fragment An embodiment of a method for purifying POI comprising the steps of inducing C-terminal protein cleavage of the protein and thereby releasing POI and separating the POI from the C-terminus of the first fusion protein and the intein C fragment.

  The present invention relates to a fusion protein comprising a desired protein (POI) and an intein C fragment, wherein the POI is fused to the C-terminus of the intein C fragment, the intein is naturally split into DnaE, and the intein C fragment is the intein C fragment. Embodiments of fusion proteins having Asp118Gly mutations within the C fragment are included.

  The present invention is a first vector comprising a first DNA element encoding the C-terminus of an intein C fragment operably linked to a promoter, the intein C fragment having an Asp118Gly mutation in the intein C fragment, A first vector having a cloning site that allows insertion of an intein C fragment of a second DNA element encoding the desired protein (POI) into the C-terminus and operably linked to a promoter; A second vector comprising a second DNA element encoding a fusion protein comprising an intein N fragment and a purification tag; or a fusion protein comprising an intein N fragment and a purification tag and a DNA element encoding POI of the first vector Instructions for insertion into the cloning site and isolation of POI And a of instructions, including an embodiment of a kit for isolating a POI.

  The present invention provides a first fusion protein comprising contacting a first fusion protein containing a desired protein (POI) fused to the C-terminus of an intein C fragment with a second fusion protein containing an intein N fragment and a purification tag. Forming a complex of the second fusion protein with a purification tag located at the intein split junction at the C-terminus of the N fragment, and cleaving POI from the intein C fragment, An embodiment of a method for purifying POI comprising the step of releasing the protein from the complex and isolating the POI.

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures.
FIG. 6 is a table with reported half-lives for various consecutive inteins and split inteins. a) The half-lives of SspDanE and split SspDnaB were calculated from pseudo-first-order kobs reported in the references. b) SspDnaE C-terminal cleavage rate after N-terminal cleavage; c) continuous intein; 2: Waugh, DS An overview of enzymatic reagents for the removal of affinity tags. Protein Expr. Purif. 80, 283-293 (2011). 3: Malakhov, MP et al. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct. Funct. Genomics 5, 75-86 (2004). 4: Mathys, S. et al. Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene 231, 1-13 (1999 ). 5: Lew, BM, Mills, KV & Paulus, H. Characteristics of protein splicing in trans mediated by a semisynthetic split intein. Biopolymers 51, 355-362 (1999). 7: Southworth, MW, Amaya, K., Evans, TC, Xu, MQ & Perler, FB Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. BioTechniques 27, 110-114, 116, 118-120 (1999); 8: Li, YF Self-cleaving fusion tags for recombinant protein production. Biotechnology Letters 33, 869-881 (2011). Diagram showing the reaction of C-GFP (1) with CBD-NpuN (2) (FIG. 2A) or CBD-NC1A (4) (FIG. 2B) at 22 ° C. in the absence or presence of DTT5 or 50 mM (12% acrylamide). CBD-GFP is a trans-splicing product and GFP is a cleaved C-extane. “+” Indicates an impurity. FIG. 5 shows a list of various fusion protein constructs used. The construct includes the engineered intein pair for characterization purposes as well as the desired protein used for sample purification. It is the figure which showed that the trans-splicing activity of wild type NpuDnaE intein was sulfur dependence. Reaction of CBD-N (2) and C-GFP (1) at 22 ° C. in the absence or presence of DTT 2 mM visualized on SDS-gel (12% acrylamide). CBD-GFP is a trans-splicing product. GFP is a cleaved C-extein. C and N are cleaved inteins. Trace amounts of cleaved N-extein CBD cannot be visualized on SDS-gel due to low concentration, but can be detected by Western blot (data not shown). “+” Indicates an unidentified band. It is the figure which showed the mechanism. FIG. 5A shows the intein trans-splicing mechanism. FIG. 5B shows the products obtained from different intein reactions. By mutating the last asparagine and the first cysteine to alanine, most of the intein N and C termini are cleaved, respectively. FIG. 5C shows the structural alignment of NpuDnaE intein (red, pdb: 2 keq) and mini-MtuRecA intein (yellow, pdb: 2IMZ). The conserved catalytic residues of Npu DnaE and mini-MtuRecA intein are highlighted in green and brown, respectively. FIG. 4 shows the sequence alignment of Ssp and Npu DnaE intein and mini-MtuRecA intein. ||: IN and IC cut points of DnaE intein. The active site residues shown in FIGS. 5C and 21 are highlighted in red. The Asn118 residue is highlighted in magenta. The numbers correspond to NpuDnaE residues (SEQ ID NOs: 67-69). It is the figure which showed the catalytic activity of the variant C ^* . Reaction of CBD-N (2) and C ^* -GFP (4) at 22 ° C. in the absence or presence of reducing agent DTT (FIG. 7A) or TCEP (FIG. 7B). 2N: Dimeric complex of CBD-N (2). The dimer disappears in samples treated with higher concentrations of β-mercaptoethanol and boiled for a longer time. GFP is a cleaved C-extein and C ^* is a cleaved C-intein. N is a cleaved N-intein. Cleaved N-Extein CBD cannot be visualized on SDS-gels, but can be detected by western blotting samples incubated with DTT (data not shown). “+” Indicates an unidentified band. FIG. 7C shows the time course of C ^* -GFP disappearance by C-terminal truncation at various temperatures. Error bars represent the standard deviation of 3 independent experiments. It is the figure which showed the catalytic activity of C ^* -GFP by CBD-NC1A. Reaction of C ^* -GFP (3) with CBD-NpuNC1A (4) at 22 ° C. in the absence or presence of DTT. GFP is a cleaved C-extein and NpuC ^* is a cleaved C-intein. “+” Indicates an impurity. It is a schematic diagram of the operated intein pair. FIG. 9A shows a comparison of current intein systems or tag removal designs versus conventional systems or designs. FIG. 9B shows the fusion protein before and after intein association. The intein N fragment (yellow) and C fragment (brown) were prepared from the NMR structure of NpuDnaE (PDB code: 2 keq). It is the figure which showed the characteristic of the C-terminal cleavage reaction rate of the operated intein system. FIGS. 10A and 10B are NC1A-CBD (construct 1) and C ^* -PTDH (construct 3), respectively, performed at 22 ° C. and 6 ° C. in pH 8 buffer containing 50 mM DTT, quantified using densitometric analysis. The SDS-gel (12% acrylamide) of the reaction with (continuous line) is shown. Cleaved C ^* is 4 kDa and cannot be visualized on a gel. FIG. 10C shows the C-terminal cleavage kinetics of wild type intein C-PTDH using NC1A-CBD at 22 ° C. in pH 8 buffer containing 50 mM DTT, quantified using densitometric analysis. FIG. 10D shows the time course of C / C ^* -PTDH disappearance by C-terminal truncation at various temperatures. The first 5 minutes are shown in the inset. Error bars represent the standard deviation of two independent tests. It is the figure which showed the C-terminal cleavage reaction rate on different conditions. FIG. 11A shows an SDS-gel of the reaction of NC1A-CBD (construct 12) and C ^* -PTDH (construct 6) performed at 22 ° C. under different buffer conditions. “+” Indicates an impurity. FIG. 11B shows the percent C-terminal truncation calculated under different conditions. Error bars represent the standard deviation of two independent experiments. It is the figure which showed the effect of the +1 residue of C terminal cutting | disconnection. SDS gel of reaction of NC1A-CBD and C ^* -X-GFP at pH 8 and 22 ° C. The calculated percent C-terminal cleavage of C ^* -X-GFP is shown below each lane. The upper case capital letter represents an amino acid substitution at position +1. The standard deviation for all cleavage reactions from two independent experiments is less than 7%. FIG. 2 shows protein purification using a development method called SIRP (Split Intein-mediated ultra-high speed purification of untagged proteins). FIG. 13 is a schematic diagram of a chitin-mediated chromatographic purification method. The lysate of C ^* -POI is passed through a column pre-bound with NC1A-CBD in the presence of 0.5 mM ZnCl2. After washing, the intein C-terminal cleavage reaction can be induced by adding DTT and the purified POI can be collected in the flow-through. The column is then regenerated by washing with pH 11.4 buffer and dissociating the intein complex. FIG. 14 shows the purification of PTDH (FIG. 14A) and GFP (FIG. 14B) using NC1A-CBD-chitin resin under SIRP method. SDS-PAGE analysis of purification of PTDH (left) and GFP (right). Lane 1, soluble fraction of lysate containing C ^* -PTDH / GFP; lane 2, soluble lysate flow-through; lane 3, lysate added and washed with buffer containing 0.5 mM ZnCl ₂ Lane 4, elution of C ^* -PTDH / GFP in pH 11.4 buffer; lane 5, chitin resin incubated for 30 minutes at 22 ° C. in cleavage buffer; lane 6, in cleavage buffer Chitin resin incubated at 6 ° C. for 3 hours; Lane 7, flow through after 30 minutes incubation in cleavage buffer at 22 ° C .; Lane 8, flow through after 3 hours incubation at 6 ° C .; Lane 9, target protein Chitin resin after elution. It is the figure which showed that chitin coupling | bonding NC1A-CBD can be regenerated | regenerated after refinement | purification. SDS-gel of sample collected during purification of PTDH using regenerated column, lane 1, chitin resin before cleavage; lane 2, chitin resin incubated at 22 ° C. in cleavage buffer for 30 minutes; lane 3, purification Flow-through containing PTDH. Cleaved C ^* is 4 kDa and cannot be visualized on a gel. FIG. 2 is a schematic diagram of two embodiments of a protein purification method. Method 1: A method that does not use a column. Method 2: Chromatographic based method. The symbol represents the E. coli cellular protein present in the lysate. FIG. 4 shows purification of PTDH, DsRed and GFP using engineered Npu ^* intein. FIG. 17A shows SDS-PAGE (10% acrylamide) analysis of a sample collected during purification of PTDH by reversible precipitation of ELP. Lane 1, pre-purified ELP-N (5); lane 2, soluble lysate containing C ^* -PTDH (3); mixture of samples from lane 3, lane 1 and 2; lane 4, precipitation of ELP complex Later supernatant; lanes 5 and 6, ELP-N and C ^* -PTDH mixture at t = 0 and 3h of intein reaction at 22 ° C, respectively; lane 7, ELP precipitation after 3h intein reaction; lanes 8 and 9, Supernatant containing purified PTDH after ammonium sulfate precipitation after 3 and 20 h intein reaction, respectively. An equivalent amount of protein was added to each lane. The black arrow indicates uncut C ^* -PTDH. FIG. 17B shows an image obtained over the course of DsRed purification by reversible precipitation of ELP. FIG. 17C shows SDS-PAGE (12% acrylamide) analysis of a sample collected during purification of GFP on a chitin column. Lanes 1 and 2, soluble lysate containing CBD-N (2) and C ^* -GFP (3), respectively; Lane 3, sample obtained from chitin beads after CBD-N (2) binding; Lane 4, C ^* -Sample obtained from chitin beads immediately after GFP (4) binding; Lane 5, sample 3 hours after intein reaction; Lane 6, sample obtained from chitin beads after elution of GFP; Lane 7, containing purified GFP Eluate Except for lanes 1 and 2, an equivalent amount of protein was added to each lane. FIG. 5 shows further sample purification of recombinant protein using reversible precipitation of ELP and self-cleaving C ^* . Lane 1, pre-purified ELP-N; lane 2, soluble lysate containing C ^* -POI; lane 3, mixture of samples of lanes 1 and 2; lane 4, supernatant after precipitation of ELP complex; lane 5 and 6, respectively, mixture of ELP-N and C ^* -POI at the start of the intein reaction and 3 hours after; lane 7, ELP precipitate after 3 hours of intein cleavage reaction; lanes 8 and 9, respectively, the intein cleavage reaction Supernatant containing purified POI after ammonium sulfate precipitation at time 3 and 20 hours. An equivalent amount of protein was added to each lane. Black arrows indicate uncut C ^* -POI (A) DsRed, (B) β-galactosidase (β-gal), (C) chloramphenic acetyltransferase (CAT), (D) maltose binding protein (MBP) , (E) Green fluorescent protein (GFP). It is the figure which showed the protein refinement | purification and quantification by ELP precipitation. ^a : intein reaction time at 22 ° C .; ^b : purification yield was determined from 25 mg of wet E. coli pellet. ^c : Percent recovery was estimated using SDS-PAGE densitometric analysis. ^d : Protein purified with an intein reaction time of 3 hours was used. One unit of β-galactosidase is defined as the amount of protein required to hydrolyze 1.0 μmole of ONPG per minute at 22 ° C. DTT interferes with absorption of the products of PTDH and CAT reactions and interferes with accurate activity assays for these proteins. The activity of MBP was analyzed only quantitatively. ^e : ND, not measured. ^f : Numbers in parentheses represent percent recovery based on activity assay. It is the figure which showed the ELP pull-down efficiency and purification yield which were calculated based on the activity assay with respect to the sample added to the SDS-PAGE gel in FIG. a: Pull-down efficiency = (lane 2 activity−lane 4 activity) / lane 2 activity × 100%. b: Yield (3 hours) = lane 8 activity / lane 2 activity × 100%. c: Yield (20 hours) = lane 9 activity / lane 2 activity × 100%. It is the figure which showed the residue involved in the charge relay required for C terminal cutting | disconnection. FIG. 21A is a schematic of the first step of the charge relay responsible for C-terminal asparagine cyclization. The corresponding residues in SspDnaE (orange) and NpuDnaE (black) are shown. FIG. 21B shows the structural alignment of charge relay residues in SspDnaE (orange, pdb: 1zd7) and NpuDnaE (basic color, pdb: 2 keq). Asn137 forms a hydrogen bond with Asp117 in NpuDnaE, making it less suitable for participation in charge relay. It is the figure which showed having incubated the reaction of C ^* -GFP (3) and CBD-NpuN (1) with DTT 5 mM at various temperature. The reaction was stopped by mixing the sample with SDS sample buffer and boiling for 5 minutes. FIG. 1 lists suitable DnaE inteins and the genera, species and strains from which they can be derived. It is the figure which showed N-intein and C-intein amino acid sequence (sequence number 70-87).

  The making and use of various embodiments of the present invention are discussed in detail below, but the present invention provides many applicable inventive concepts that can be implemented in a wide variety of specific situations. I want you to understand. The specific embodiments described herein are merely illustrative of specific ways to make and use the invention, and do not limit the scope of the invention.

  In order to facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” do not only mean a singular entity, but also a specific example is used for illustration. Including its general type that can. The terminology herein is used to describe specific embodiments of the invention, but their use does not limit the invention except as outlined in the claims. .

  The term “gene” is used to mean a unit that encodes a functional protein, polypeptide or peptide. As will be appreciated by those skilled in the art, this functional term includes any of genomic products, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that can be modified by human hands. . Purified genes, nucleic acids, proteins, etc. are usually used to mean these entities when identified and separated from at least one contaminating nucleic acid or protein of interest.

  As used herein, the term “vector” is used in reference to nucleic acid molecules that transfer DNA fragments from one cell to another. A vector is further defined as a vector designed to increase a specific sequence, or an expression vector containing a promoter operably linked to a specific sequence, or a vector designed to cause the introduction of such a promoter. be able to. The vector may exist independently of the host cell chromosome or may be integrated into the host cell chromosome.

  The term “host cell” means a cell that has been engineered to contain a nucleic acid segment or a modified segment, whether archaea, prokaryotic or eukaryotic. Thus, engineered or recombined cells can be distinguished from naturally occurring cells that do not contain a transgene that has been recombined by the hand of man.

  With respect to nucleic acid or polypeptide sequences, the term “altered” or “altered” or “altered” refers to changes such as insertions, deletions, substitutions, fusions with related or unrelated sequences, It is meant to include changes that may occur by hand, or naturally occurring changes such as polymorphisms, alleles and other structural types. Modifications include genomic DNA and RNA sequences that differ in their hybridization properties using a given hybridization probe. Modifications of the polynucleotide sequence or fragments thereof include those that increase, decrease or have no effect on function. Polypeptide alteration means amino acid derivatives or conjugates, or those altered by recombinant DNA manipulation such as post-translational modification, chemical or biochemical modification.

  The term “control sequence” refers to a DNA sequence that is required for expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and a transcription terminator. A strongly regulated inducible promoter can be used that suppresses Fab 'polypeptide synthesis at a level below the growth inhibitory amount during cell culture growth and maturation, for example, during log phase.

  A nucleic acid is “operably linked” when it is functionally related to another nucleic acid sequence. For example, when expressed as a protein precursor involved in polypeptide secretion, a presequence or secretory leader DNA is operably linked to the polypeptide DNA to achieve sequence transcription, coding a promoter or enhancer. A ribosome binding site is operably linked to a coding sequence when operably linked to the sequence or arranged to facilitate translation. In general, “operably linked” means that the bound DNA sequences are contiguous and, in the case of a secretory leader, are contiguous within the same reading frame. Enhancers do not have to be contiguous. Binding is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

  By “exogenous” element is meant herein a nucleic acid sequence that is foreign to the cell or is a homologue to the cell but in a position within the host cell nucleic acid that is not normally found. Is defined as

  As used herein, the expressions “cell” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the terms “transformants” and “transformed cells” include the primary subject cell and cultures obtained from them regardless of the number of passages. It should be understood that all progeny may not have exactly matched DNA contents due to planned or accidental mutations. Variant progeny that have the same function or biological activity as screened on the original transformed cells are included. Various names will be clear from the context.

  A “plasmid” is designated by uppercase letters and / or lowercase letters p before and after numbers. The starting plasmids herein are commercially available and are publicly available without limitation, or can be constructed from such available plasmids according to published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to those skilled in the art.

  “Recovery” or “isolation” of a given fragment of DNA from restriction enzyme digestion refers to the separation of the digest on polyacrylamide or agarose gel by electrophoresis, its mobility relative to a marker DNA fragment of known molecular weight. It means identification of the desired fragment by comparison, removal of the gel part containing the desired fragment, and separation of the gel from DNA. This technique is generally known. See, for example, Lawn et al. (Nucleic Acids Res. 1981. 9: 6103-6114) and Goeddel et al. (Nucleic Acids Res. 1980. 8: 4057).

  “Preparation” of DNA from cells means the isolation of plasmid DNA from a culture of host cells. Commonly used methods for DNA preparation are the large and small plasmid preparations described in Sections 1.25 to 1.33 of Sambrook et al., (Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory Press, 1989). It is. The DNA preparation is purified by methods well known in the art (see Sambrook et al., Section 1.40 above).

  As used herein, the term “protein-protein complex” or “protein complex” means an association of one or more proteins. The proteins of the complex can be associated by various means or any combination of means including but not limited to functional, stereochemical, structural, biochemical or electrostatic association. The term is intended to encompass the association of any number of proteins.

  As used herein, the term “protein”, “polypeptide” or “peptide” means a compound comprising amino acids joined by peptide bonds and is used interchangeably.

  The term “desired protein” as used herein means the protein, its function and / or expression that it is desired to isolate or purify using the methods and constructs of the invention. The present invention may be useful for the isolation and / or purification of any protein expressed by any gene of any organism, whether prokaryotic or eukaryotic.

  Regarding nucleotides, the terms “a sequence essentially as set forth in SEQ ID NO. (#)”, “A sequence similar to”, “nucleotide sequence “Nucleotide sequence” and like terms means a sequence that substantially corresponds to any portion of the sequence identified herein as SEQ ID NO: 1. These terms refer to molecules obtained synthetically as well as naturally occurring, eg, biological, immunological, experimental, with respect to hybridization with nucleic acid sequences, or the ability to encode all or part of an activity. Or other sequences having functionally equivalent activity. Of course, these terms are meant to include such sequence information specified by its linear order.

  The term “homology” refers to the extent to which two nucleic acids are complementary. There may be partial or complete homology. A partially complementary sequence is a sequence that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid and is expressed using the functional term “substantially homologous”. The The degree or range of hybridization can be examined using hybridization or other assays (competitive PCR assays), etc., and as specifically known to those skilled in the art, it interacts specifically even with low stringency. Is included.

  Inhibition of hybridization of sequences that are fully complementary to the target sequence also uses hybridization assays involving solid supports (eg, Southern or Northern blots, solution hybridizations, etc.) under conditions of low stringency. Can be examined. Low stringency conditions can be used to identify that two sequences bind to each other and are still specific (ie, selective). The absence of non-specific binding can be tested by using a second target that lacks even partial complementarity (eg, below about 30% identity). Without non-specific binding, the probe will not hybridize to the second non-complementary target and the original interaction is found to be selective. Low stringency conditions are generally 5 × SSPE (NaCl 43.8 g / l, NaH 2 PO 4 —H 2 O 6.9 g / l and EDTA 1.85 g / l, pH 7.4), 0.1% SDS, 5 × Denhart Binding at 42 degrees Celsius in a solution consisting of 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma) and 100 microgram / ml denatured sperm DNA per 500 ml. Alternatively, when a probe having a length of about 500 nucleotides is used under the same conditions as hybridization, the probe is subsequently washed with a solution containing 5 × SSPE and 0.1% SDS at 42 degrees Celsius. It is well known in the art that many equivalent conditions can be used to achieve low stringency conditions. Factors that affect the level of stringency include probe length and nature (DNA, RNA, base composition) and target nature (DNA, RNA, base composition, whether present in solution or immobilized Etc.) and salts and other components (eg, formamide, dextran sulfate, polyethylene glycol). Similarly, the hybridization solution can be varied to produce conditions of low stringency hybridization that differ from those described above but are equivalent. Furthermore, conditions that promote hybridization under conditions of high stringency (eg, increased hybridization temperature and / or increased wash steps, inclusion of formamide, etc.) are known in the art.

  We have developed an ultra-fast method (SIRP) that allows the use of engineered split inteins to purify untagged proteins in a short time. In certain embodiments, this technology provides a powerful new method for purifying untagged proteins in small scale applications. In certain embodiments, low cost chitin beads are used. In other embodiments, a low cost CBD-bound amorphous cellulose matrix is used as the affinity support for CBD. This makes the use of this intein-mediated method attractive as an affinity-based step for large-scale protein purification. In other embodiments, precipitation of elastin-like polypeptide (ELP) is used.

  Some embodiments include the inteins listed in FIGS. 23 and 24.

  We recognize that rapid and efficient removal of tags is an important issue in the purification of recombinant proteins. Using engineered C-terminal truncation of the natural split DnaE intein of Nostock punctiforme, we can purify untagged recombinant proteins from E. coli lysates in less than 1 hour. An ultra-rapid purification (SIRP) of split-tained untagged proteins has been developed.

  We recognize that affinity tags simplify the purification of recombinant proteins and are extremely important for modern biotechnology. However, there is currently no simple and low-cost method for removing affinity tags, so the protease required for tag removal takes extra time and high costs, so in large industrial processes Usefulness is greatly impaired. Most proteases used for tag removal have low specificity or low activity and liberate specific amino acids of the target protein after cleavage. The recently discovered SUMO-protease shows both high specificity and efficiency (over 90% complete within 20 minutes at 22 ° C.). However, the cost of cleaving 1 g of recombinant protein using SUMO-protease (Life Technologies) is $ 673,000 and cannot be used in most applications.

  The present inventors have recognized that a protease purification method has been developed that uses inteins that have been engineered to perform N / C terminal cleavage reactions in an acidic (pH 6) or reducing environment. Inteins are proteins that catalyze a splicing reaction that binds related N- and C-extanes via peptide bonds. Inteins can be engineered to perform a single cleavage reaction at the N- or C-terminus in an acidic (pH 6) or reducing environment, and these engineered inteins can be labeled with stimulus-responsive tags in protein purification applications. Can be used to remove. However, the engineered inteins used in these protein purification applications are always inefficient and need to be incubated at least overnight to achieve significant cleavage / tag removal (FIG. 1). Also in these systems, cleavage is incomplete in vivo and the target protein is released. Thus, unless slow-acting inteins are used, the conditions for expressing the target protein often need to be optimized to minimize intein cleavage in vivo. In some cases, even after optimizing the expression conditions, incomplete protein cleavage still significantly affects the target protein yield. We carry out the cleavage / tag removal reaction in a much shorter time without the possibility of inducing incomplete cleavage by inteins in vivo, and thus, economical, widely used, tagged An intein-based system for the purification of non-recombinant proteins was created.

  In certain embodiments, we use split inteins that split their catalytic residues between two peptide chains: an N-terminal intein (IN) and a C-terminal intein (IC) to target in vivo Recognizes solutions that prevent protein cleavage. Split inteins are active only when two fragments are associated. Two protein purification systems were developed using artificially split DnaB inteins of Synethocystis sp. (Ssp). In one, the artificial split S1 DnaB intein consists of 11-amino acid N-intein (IN) and 144-amino acid C-intein (IC). The target protein is fused to the IC and tag removal is achieved by adding an IN peptide. Since there is no N-extane, the wild type catalytic residue is retained. In order to achieve sufficient cleavage, a target protein fused with IN to IC in a 40 to 1 molar ratio is required. Despite the small size of IN, peptide synthesis is expensive and this system cannot be used in large scale processes. A similar affinity-based purification system using the same SspDnaB intein with different split junctions was also developed. In this system, mutations in the appropriate catalytic residues at the N and C terminus are used to achieve C and N terminal truncation, respectively. However, despite the relatively fast reaction rate of wild-type SspDnaB (FIG. 1), the mutant intein has a slow reaction rate and it is necessary to extend the incubation time at room temperature to achieve sufficient C-terminal cleavage. Yes (16 hours). The usefulness of this system is limited due to the long incubation time.

  In certain embodiments, we have two additional restrictions on the use of artificial split inteins: artificial split inteins are (1) partially compatible with split fragments Since it is low, the activity is lower than the continuous one, and (2) it is recognized that when expressed alone, the tendency to form aggregates is high. We recognize that natural split inteins such as Ssp and Npu's DnaE are highly active, soluble and exhibit very high affinity between the two split fragments. Neither of the two natural split DnaE inteins has been used for protein purification. The highly exposed hydrophobic surface of DnaE IN (Npu and Ssp, DnaE 102 and 123 amino acids, respectively) tends to interfere with the folding of N-extane, and some fusion proteins are misfolded Cause the formation of insoluble aggregates, limiting the usefulness of N fragments as a general purification tag. Although we have a small size (both Npu and Ssp are 36 amino acids) and do not significantly interfere with the solubility of the target protein, the C fragment of DnaE is also closely related to the C and N-terminal cleavage reactions. It is recognized that it is not suitable as a purification tag. In natural split DnaE inteins, C-terminal cleavage can only occur after N-terminal cleavage, and mutating the first Cys to Ala normally prevents N-terminal cleavage without interfering with C-terminal cleavage activity, C-terminal truncation is also eliminated (FIG. 2B).

For certain embodiments, we introduce a single mutation, Asp118, based on sequence alignment to the mini-MtuRecA intein to cut the C-terminus without cutting the N-terminus. NpuDnaE (referred to as Npu ^* ) was manipulated. Npu ^* achieves a C-terminal cleavage yield of about 80% within 3 hours of reaction at 22 ° C. For comparison, to achieve the same degree of C-terminal cleavage, the IMPACT system (New England Biolab) using SceVma1 and SspDnaB intein (IMPACT Manual) takes about 16 hours at 23 ° C. Using Npu ^*, we have further developed two protein purification methods that are electrophoretically in high yield (up to 84 mg per liter of E. coli culture) within a short time (less than 4 hours). Purely purifying multiple target proteins suggested the usefulness of these techniques and the potential for large-scale industrial protein purification.

  We disclose the manipulation of DnaE inteins that can catalyze rapid C-terminal cleavage in the absence of N-terminal cleavage. In a specific embodiment, a single mutation, Asp118Gly, was introduced into the DnaE intein (NpuDnaE) of Nostock punctiforme PCC73102, based on a sequence alignment with a previously engineered C-terminally truncated intein mini-MtuRecA. This mutation could suppress trans-splicin activity, delay N-terminal cleavage and significantly increase C-terminal cleavage efficiency. Molecular modeling suggests that in NpuDnaE, Asp118 forms a hydrogen bond with the penultimate Asn, preventing spontaneous cyclization prior to N-terminal cleavage. Mutating Asp118 to Gly removes this restriction, which then leads to C-terminal truncation in the absence of N-terminal truncation. The Gly118 NpuDnaE mutant shows a rapid sulfur-dependent (or sulfur-induced) C-terminal cleavage reaction rate, which is 80% complete within 3 hours at room temperature. In various embodiments, we have used this newly engineered intein as a removable purification tag for protein purification methods that do not use a column that utilizes an elastin-like peptide and a chromatography that utilizes a chitin binding protein. Both methods of protein purification based on graphy were developed. In certain embodiments, the target protein is rapidly purified with electrophoretic purity and a yield of up to 84 mg per liter of E. coli culture.

  In certain embodiments, various fusion proteins containing NpuDnaE IN or IC (N or C) were generated as illustrated in FIG. 3 to analyze the in vitro activity of inteins. For application as a protein purification tag, it is important that intein activity is regulated by external stimuli. Theoretically, the intein reaction does not require any thiol reagent, but an unpaired cysteine can form an intramolecular disulfide bond within the NpuDnaE intein and can interfere with the intein reaction by forming a redox trap. There are several residues. We measured whether NpuDnaE activity was sulfur dependent. Purified CBD-N (construct 2) was mixed with the same concentration of C-GFP (construct 1) in the absence or presence of 2 mM DTT. Since the trans-splicing product CBD-GFP was present only in reactions containing DTT, it was confirmed that split NpuDnaE intein is sulfur dependent (FIG. 4). The trans-splicing reaction is almost complete after 30 minutes. Trace amounts of cleaved N and C exteins can also be visualized and are somewhat noticeable in reactions performed at a DTT concentration of 50 mM (FIG. 2A). DTT can initiate a nucleophilic attack on the thioester bond of a linear or branched intermediate (FIG. 5A, steps 1 and 2, respectively), causing an N-terminal cleavage. We recognize that with SspDnaE intein, the presence of DTT 50 mM has found that protein trans-splicing is almost completely blocked and the N-terminal cleavage and subsequent C-terminal cleavage reactions are blocked. . Even at DTT 50 mM, the limited amount of N-terminal truncation observed in the NpuDnaE intein is very significant if the conversion of linear / branched intermediates into trans-splicing products effectively competes with nucleophilic attack by DTT. This may be due to rapid trans-splicing kinetics.

  In a specific embodiment, mutant N, CBD-NC1A (construct 4), in which the first Cys is replaced with Ala, does not show trans-splicing activity even at DTT 50 mM, C-terminal cleavage activity is negligible (FIG. 2B), The cleavage reaction was consistent with previous findings that it was closely coupled with N-terminal cleavage in DnaE intein. We recognize that this unique property precludes the use of wild-type DnaE inteins for C-terminal protein purification applications.

  Rational design of Npu DnaE that cleaves the C-terminus: With some exceptions, most intein splicing reactions involve four highly coordinated nucleophilic substitutions (FIG. 5A). The first step involves an NX acyl shift (X: C or S) where the first residue of the intein, cysteine (Cys1) or serine attacks the previous peptide carbonyl to form a linear (thio) ester. . In the second step, the first residue of C-extein, cysteine (Cys + 1), serine or threonine attacks this (thio) ester carbonyl and cleaves N extein, one belonging to intein The other forms a branched intermediate with two N-termini belonging to the N extein. In this situation, the exteins bind together but are still bound to the intein C-terminus. This branched intermediate is then cleaved from the intein by a transamidation reaction involving the last asparagine residue of the intein (step 3). In the final step, the free extein undergoes a spontaneous X-N acyl shift, reverting the (thio) ester to the peptide bond and forming a spliced protein product.

  In most inteins, the reactions at the N and C termini are independent, and thus mutation of the catalytic residue that eliminates the reaction at one terminus causes a cleavage reaction at the other terminus (FIGS. 1 and 5B). However, conventional methods cannot be used to manipulate C-terminal truncated DnaE inteins because the N and C terminal cleavage reactions are closely conjugated. The C-terminal truncated intein, mini-MtuRecA, was engineered using directed evolution. It was discovered that a single mutation, D422G, is responsible for increasing C-terminal cleavage activity and suppressing N-terminal cleavage. Alignment of NpuDnaE and mini-MtuRecA intein revealed high sequence level homology (FIG. 6) and higher structural level homology (FIG. 5C). Most of the catalytic residues including Asp422 (NspDnaE Asp118) are conserved between NpuDnaE and the mini-MtuRecA intein (FIG. 6). We recognize that mutation D118G can confer C-terminal cleavage activity to NpuDnaE intein.

Activity of Npu DnaE intein with Asp118Gly mutation: To test the effect of D118G mutation, amino acid substitutions were introduced into C-GFP via site-directed mutagenesis to form C ^* -GFP (construct 3). Similar to wild type NpuDnaE, the activity of mutant Npu ^* is also sulfur dependent. The D118G mutation completely abolished the trans-splicing reaction and induced rapid C-terminal truncation under reducing conditions (FIG. 7A). Spontaneous C-terminal cleavage was not observed after 20 hours when C ^* -GFP alone or with DTT was incubated at room temperature, confirming that C-terminal cleavage activity is N-dependent. When the reaction was carried out at a low DTT concentration, almost no free N-extein was observed, and in the reaction at a high DTT concentration (50 mM), only a very limited amount of free N-extein was observed. Was suggested to essentially eliminate the first NX acyl shift and induce a C-terminal cleavage reaction independent of N-terminal cleavage. To further confirm that Npu ^* can perform C-terminal cleavage in the absence of N-terminal cleavage, we have determined that trialkylphosphines can break disulfide bonds but not thioester bonds. The reaction was carried out in the presence of (tris (2-carboxyethyl) phosphone, TCEP). TCEP also induces a C-terminal cleavage reaction without any N-terminal cleavage (FIG. 7B), suggesting that Npu ^* is not coupled with N- and C-terminal cleavage activities. We also measured the activity of C ^* -GFP when mixed with CBD-NC1A (construct 4). A similar rate of C-terminal cleavage was observed under reducing conditions. However, rapid C-terminal truncation was observed even in the absence of DTT, making it inappropriate for use as a controllable protein purification tag (FIG. 8). Cys1 and Cys + 1 are so close that they can form a disulfide bond immediately adjacent to the association of the two intein fragments, further hindering the intein reaction and allowing control of the initiation of C-terminal cleavage by reducing agents. To. We also measured the Npu ^* C-terminal cleavage kinetics at various temperatures (FIG. 7C). The highest cutting speed was obtained at 37 ° C. and about 80% cutting was achieved in just 1 hour. Samples incubated at room temperature (22 ° C) required 3 hours and 4.5 hours at 16 ° C to achieve the same 80% cleavage. More than 85% C-terminal truncation was obtained after 20 hours at 4 ° C. This cleavage rate is significantly faster than that of the SspDnaB and SceVma1 inteins currently used in the IMPACT system (New England Biolab) which requires about 16 hours incubation at 23 ° C. to achieve similar cleavage efficiency. . If N is present in excess, it may be possible to achieve faster cutting speeds. Taken together, these results suggest the usefulness of Npu ^* as a self-cleaving tag for protein purification.

  Abbreviations: ELP, elastin-like peptide; CBD chitin binding domain; POI, desired protein; Mtu, Mycobacterium tuberculosis; Npu, Nostock punctiforme; IN / IC, split intein N / C fragment; N / C, NpuDnaE N / C fragment; IPTG, isopropyl β-D-1-thiogalactopyranoside; SDS, sodium dodecyl sulfate.

In order to obtain the C-terminal cleavage kinetics and efficiency used in SIRP, we rearranged the protein purification tag at the intein split junction (C-terminus of the intein N fragment) and placed the target protein at the C-terminus of the C fragment. Fused to. This system showed extremely rapid sulfur-induced C-terminal truncation and was about 50% complete within 30 seconds at both 22 ° C. and 6 ° C. This is the fastest C-terminal cleavage activity reported to date for inteins. Although the reaction rate seemed to slow down after the first minute, more than 85% cleavage completion was achieved within 30 minutes at 22 ° C and within 3 hours at 6 ° C. Ultrafast cleavage kinetics are made possible by placing the purification tag at the intein split junction, thus avoiding the possibility of steric hindrance of important interactions between N- and C-exteins. The C-terminal cleavage efficiency of the engineered split intein is not affected by what is the first residue of the C-extane (proline was found to be an exception), and the purified tag was completely removed by SIRP. It was possible to remove and purify the protein with the natural N-terminus. The C-terminal cleavage reaction can be effectively inhibited by divalent Zn ²⁺ under non-reducing conditions. Importantly, because the association of intein N and C fragments is reversible, the column-bound intein N fragment that captures the protein can be regenerated for multiple uses, further reducing the cost of protein purification. SIRP technology should provide a useful means for purifying untagged proteins and peptides.

The natural split DnaE (NpuDnaE) of Nostock punctiforme has very high trans-splicing activity and does not produce incomplete intein cleavage in vivo because the constituent fragments are expressed in separate hosts. We engineered NpuDnaE to perform sulfur-induced C-terminal truncation by introducing the point mutation, Asp118Gly, into the C-intein fragment (C) to produce mutant C ^* . In certain embodiments, the first residue (N) of the N-intein fragment is mutated to Ala (NC1A) to completely eliminate any N-terminal cleavage activity, and the affinity tag, chitin binding domain (CBD) Was added to the C-terminus of NC1A to generate construct NC1A-CBD (construct 12). This construct contains 1 Met as N-extein, uses affinity tag as N-extein and is used for protein purification that can interfere with cleavage activity by steric hindrance by C-extein This is in contrast to the conventional intein system (FIG. 9A). The desired protein (POI) was attached to the C-terminus of C ^* .

We have made various fusion proteins containing engineered intein pairs as listed in FIG. To measure the C-terminal cleavage kinetics, ^C * -PTDH comprising ^{C *} fused to globular proteins phosphite dehydrogenase (PTDH) a (construct 6), NC1A-CBD and (construct 12) 1: 1 mole The ratio was mixed in the presence of 50 mM DTT. Approximately 50% of PTDH was cleaved from C ^* -PTDH within 30 seconds at both 22 ° C. and 6 ° C. (FIGS. 10, A and B). Since 30 seconds is the earliest time that can be accurately measured, the time required to achieve such a cut may be even shorter. In contrast, the fastest time reported for the C-terminal truncated intein, gp41-1C1A, is t _1/2 5 minutes at 37 ° C. The reaction rate slows down in the first minute, but over 85% C-terminal cleavage is achieved within 30 minutes at 22 ° C. and within 3 hours at 6 ° C. (FIG. 10D). This rapid cleavage rate is probably due to the elimination of 1) high activity of wild-type NpuDnaE, 2) the mutated Asp118Gly in the intein C fragment, and 3) N-extein that may be interfered with POI. Interestingly, unlike CBD-NC1A (construct 4), which cannot be used to cleave wild-type C, NC1A-CBD can also induce significant C-terminal truncation of wild-type C upon association, 3 About 30% is cut in time (FIGS. 10D, 2B). These data indicate that the C-terminal truncation is severely limited until the N-terminal truncation is seen with the NpuDnaE intein, as is also observed with the SspDnaE intein, due at least in part to the presence of N-extane. Is further demonstrated.

NC1A-CBD also induces rapid C-terminal cleavage of C ^* -PTDH under non-reducing conditions, although at a much slower rate than under reducing conditions (FIG. 11B, Zn2 + 0 mM). Even in the absence of DDT, more than 50% of C ^* -PTDH is cleaved within 30 minutes at both neutral and acidic pH. This basic level of cleavage is probably due to the absence of Cys1, which can form a disulfide bond with Cys + 1 and inhibit the intein reaction. We tested the ability of Zn ²⁺ to inhibit C-terminal truncation of engineered NpuDnaE constructs. As shown in FIG. 11A, ZnCl ₂ (0.5 mM) effectively inhibits the C-terminal cleavage reaction under non-reducing conditions, but has little inhibitory effect in the presence of DTT. Inhibition is more potent at pH 6, with only about 10% intein cleavage after 3 hours incubation at 22 ° C. These results demonstrate that Zn ²⁺ and DTT can be used as effective switches for C-terminal truncation termination and initiation, respectively. Higher Zn ²⁺ concentrations can more efficiently inhibit the C-terminal cleavage reaction and help prevent product loss during long washing steps. We recognize that Zn ²⁺ ions at concentrations of 1 mM and above may precipitate some cellular proteins and therefore should not be used directly in cell lysates. According to the crystal structure of SspDnaE, Zn ²⁺ coordinates to Asp140, His48 (equivalent to Asp118, His48 of NpuDnaE) and Cys + 116. However, when Asp118 is mutated to Gly in C ^* , C-terminal cleavage activity is conferred in the absence of N-terminal cleavage. Therefore, we recognize that there is another site for Zn ²⁺ binding in NpuDnaE. We also recognize that other ions / molecules constitute cleavage inhibitors and can inhibit C-terminal cleavage more effectively than Zn ²⁺ under non-reducing conditions.

In certain embodiments, it is desirable to completely remove all unnatural amino acids from the target protein. For the intein trans-splicing reaction, a cysteine at position +1 is required to complete the transesterification and S / O-N acyl shift reactions. However, Cys + 1 is not required for the asparagine cyclization reaction involved in C-terminal cleavage. We have five different amino acids, Ala, as representative of side-chain amino acids that are small, large, hydrophobic, polar, positive and negatively charged in the first residue (X) of C-extein. Various C ^* -X-GFP fusion proteins (FIG. 3, constructs 14-18) were designed that replaced Leu, Asp, Arg and Pro. All other substitutions that occurred, except Pro + 1, completed C-terminal cleavage after 30 minutes at room temperature (FIG. 12) and had a cleavage profile similar to that seen with the original C ^* construct containing Cys at position +1 (FIG. 10).

Certain embodiments include a chitin binding domain (CBD), and to demonstrate the utility of engineered intein pairs, we designed a protein purification method based on the chitin binding domain (CBD) (FIG. 13) Two proteins were purified by chitin affinity chromatography (FIGS. 14A and 14B). 14 mg of high-purity PTDH and 18 mg of GFP were obtained per mL of chitin resin. The molar ratio of the binding of ^C * -PTDH and ^C * -GFP to NC1A-CBD, as measured by SDS-PAGE analysis, 0.35 and 0.92, respectively. The difference in binding ability is probably due to the large size of the dimeric PTDH compared to the globular single domain GFP. The intein C-terminal cleavage efficiencies of both proteins when immobilized on an affinity resin are similar to those observed in solution, with more than 80% cleaving at 22 ° C. for 30 minutes and 6 ° C. for 3 hours (see FIG. 14, lanes 5 and 6). There is a small amount of cleaved GFP in the C ^* -GFP sample (FIG. 14B, lane 3). This is mainly due to proteolytic cleavage during cell lysis and non-specific interaction between GFP and chitin resin. Since uncleaved C ^* -PTDH / GFP elutes from chitin-bound NC1A-CBD selectively with pH 11.4 buffer, the association of C ^* and NC1A is reversible (FIG. 14, lane 4), C ^* and NC1A It demonstrates that the high affinity between is largely determined by electrostatic interactions.

In certain embodiments, a fusion protein comprising NC1A is reused. In order to demonstrate the reusability of chitin-bound NC1A-CBD, we repeated the purification of PTDH using the same chitin column four times (FIG. 15). In order to remove the cleaved C ^* from the NC1A-CBD on the column after elution of the cleaved PTDH and before adding the fresh lysate containing C ^* -PTDH, the chitin resin was pH 11.4 buffered. Wash thoroughly. The yield of purified PTDH was similar in all four cycles, confirming that the NC1A-CBD-chitin affinity matrix was capable of regeneration for multiple use cycles. Cut ^{C *} It is clear can be dissociated from NC1A-CBD easily in pH11.4 buffer than the full length ^C * -PTDH.

We prefer to use DTT as a cleavage inducer for protein elution for proteins that utilize disulfide bonds exposed to the surface for certain applications, such as 3D and 4D structures. Recognize that there is no. The inventors recognize that EDTA can be used as an inducer of C-terminal cleavage if it can chelate Zn ²⁺ ions that inhibit basic cleavage and liberate POI (FIG. 11B, DTT and Zn ²⁺ 0 mM).

Reversible precipitation and protein purification with chitin resin: To demonstrate the utility of Npu ^* in protein purification, we developed various embodiments of protein purification methods (FIG. 16). Certain embodiments include a method that combines reversible precipitation of elastin-like peptide (ELP) with controllable Npu ^* C-terminal truncation (FIG. 16, Method 1). C ^* -POI in which N is attached to the C-terminus of elastin-like polypeptide (ELP) via the mobile linker ELP-N (construct 5) and the mutant C ^* is fused to the N-terminus of various sample target proteins (Construct 3, 6-10). Under non-reducing conditions, N and C ^* interact non-covalently with each other without cleavage, causing the POI and ELP to physically associate. Addition of ammonium sulfate induces ELP phase separation and causes aggregation of ELP and associated POI. After removal of cellular proteins in the supernatant, the precipitate is then lysed in low salt reduction buffer, reversing the phase transition and inducing C-terminal cleavage of the intein. Upon cleavage, POI is released from the ELP-intein complex and is selectively removed by a second ammonium sulfate precipitation and centrifugation, resulting in a highly pure POI in solution.

  An example of purification of globular protein phosphite dehydrogenase (PTDH) is shown in FIG. 17A. The yield from the intein self-cleavage reaction was 49.8 and 59.3 mg of purified PTDH per liter of E. coli culture at 22 ° C. for 3 hours and 20 hours, respectively. Purification of five additional proteins of various sizes and multimers (constructs 3, 7-10) are shown in FIGS. The purification method for DsRed (construct 7) can be conveniently monitored by visual inspection (FIG. 17B). The target protein purification yield and percent recovery from the soluble lysate are summarized in FIG. High purity was obtained for all proteins tested. Not surprisingly, the ELP reduction efficiency and intein cleavage kinetics are affected by the target protein (FIG. 20). Both monomeric and multimeric proteins can be efficiently purified by this method. In most cases, the intein self-cleavage reaction is essentially complete in 4 hours, and the time to complete the entire procedure is comparable to conventional chromatographic protein purification methods.

  Since chromatographic-based methods are still the pillars of recombinant protein purification, we also implement using affinity-based purification methods to further expand the use of engineered inteins. Developed form. In this method, ELP is replaced with a chitin binding domain (CBD) and purification is performed by binding to chitin beads (FIG. 16, method 2). An example of GFP purification is shown in FIG. 17C. The yield of purified GFP was about 2 mg on a 100 μl chitin column. The binding capacity of chitin beads appears to be much higher than that reported for maltose binding protein (0.2 mg / 100 μL of chitin beads, New England Biolabs website). The exact reason is not known, but may be due in part to the much smaller size of N compared to maltose binding protein.

Certain embodiments include engineered split NpuDnaE inteins that can perform rapid C-terminal cleavage reactions without cleaving the N-terminus. Split NpuDnaE intein is one of the most active inteins identified today (FIG. 1). However, despite the rapid reaction rate and high solubility of NpuDnaE, DnaE inteins cannot be used in many applications such as protein purification because the C-terminal asparagine cyclization relies on the N-terminal acyl shift. Although the multi-step catalytic pathway leading to intein trans-splicing is closely coordinated, the exact mechanism involved in this series of reactions remains unknown. Intein N / C end truncation can result from an increase in the cleavage rate at the splice junction or a decrease in the reaction rate of another step. The inventors have recognized that a single mutation in the mini-MtuRecA intein, D422G, can eliminate trans-splicing activity and significantly increase C-terminal cleavage activity. Asp422 is in a conserved block F region (also referred to as the C2 motif) within the intein active site and is 75% conserved among all inteins of various species including split NpuDnaE and SspDnaE inteins (FIG. 6). This block F aspartic acid has previously been shown to be essential for both the first and second steps of the intein reaction (FIG. 5A). The crystal structures of multiple inteins, including SspDnaE, show that this aspartic acid forms a hydrogen bond with the oxyanion of the N-terminal Cys1, and this residue is located for the first step NX acyl shift. It seems. Furthermore, quantum mechanical simulation and structural studies of other inteins suggest that this aspartic acid can also deprotonate the Cys + 1 thiol group and initiate nucleophilic attack to form branched intermediates. ing. Mutation of this Asp422 in mini-MtuRecA significantly slows the N-terminal cleavage reaction and the formation of branched intermediates. In the NMR structure, this position contained Ala, but in the NMR structure of NpuDnaE intein, Asp118 (equivalent to Asp422 of mini-MtuRecA) is within the hydrogen bond distance with the oxyanion of the first residue. Showed that. Npu ^* whose corresponding aspartic acid was changed to Gly showed very limited N-terminal truncation even at DTT 50 mM (FIG. 7A), and this mutation was also probably due to the first step NX acyl shift and branched intermediates. Suggests blocking formation.

In the mini-MtuRecA intein, the C and N-terminal cleavage reactions are not conjugated, and therefore the delay in the first and second steps of the intein reaction causes the C-terminal cleavage product to rise. However, with DnaE intein, the C and N-terminal cleavage reactions are strongly conjugated. Mutant DnaE inteins with C1A show little or no C-terminal truncation, so inhibiting the first two steps does not directly lead to an increase in C-terminal truncation products. To understand how D118G induces C-terminal truncation in the NpuDnaE intein, we compared the solution structure of NpuDnaE with the crystal structure of its closest homolog SspDnaE. In the SspDnaE intein, C-terminal asparagine cyclization is mediated by a charge relay process involving His147, Asn159, Arg73 and water molecules near the C-terminal splicing junction (FIG. 21A). The water molecules are within the hydrogen bond distance of the Asn159 and His147 Nδ atoms and the main chain nitrogen of Leu143, transferring protons from Asn159 to His147 (FIG. 21A). Deprotonated Asn159 Nδ initiates nucleophilic attack on its carbonyl carbon atom, causing cleavage of the C-terminal intein-extein bond. The same mechanism is involved in asparagine cyclization in SspDnaB and mini-MtuRecA containing water molecules at similar positions. The NMR structure of NpuDnaE shows that Asp118 can form a hydrogen bond with Nδ of Asn137, making it inconvenient for charge relay and thus inhibiting C-terminal cleavage (FIG. 21B). In some studies, the formation of a branched intermediate that was shown to require protonation of Asp118 by the hydrogen of the Cys + 1 thiol group disrupted the H-bond interaction of Asn137 and Asp118, resulting in charge relaying. Asn137 is involved in leading C-terminal truncation. Formation of the branched intermediate causes a slight change in the intein structure that accelerates the C-terminal asparagine cyclization reaction. Mutating Asp118 to a smaller Gly eliminates H-bond interactions and allows Asn137 to participate freely in charge relays without the need for structural changes, leading to uncoupled C-terminal truncation. Thus, in addition to inhibiting the first two steps of the intein reaction, D118G can also accelerate asparagine cyclization at Npu ^* , leading to rapid C-terminal truncation. FIG. 22 shows that the reaction of C ^* -GFP (3) and CBD-NpuN (1) was incubated with 5 mM DTT at various temperatures. The reaction was stopped by mixing the sample with SDS sample buffer and boiling for 5 minutes.

The Npu ^* cleavage rate is slightly slower than the wild-type intein trans-splicing reaction (FIG. 1). This may be due to the incomplete arrangement of Asn137 in the absence of the Asp118 side chain. Nevertheless, about 80% C-terminal cleavage can be achieved within 3 hours at room temperature, and this mutant intein is beneficial for tag removal in protein purification. Using Npu ^*, we developed a column-free protein purification method and a chromatography-based protein purification method by substituting ELP and CBD for N-extein, respectively. Various target proteins of various sizes and multimers were shown to be rapidly purified (less than 4 hours) in high purity and yield (Figure 19, 17A and 18A-18E). However, since a reducing agent is used to induce intein cleavage, this method was not tested in the purification of proteins containing naturally occurring disulfide bonds. In the first method, the use of ELP should eliminate the need for costly columns and facilitate its use in large-scale industrial protein purification. In the second method, it is convenient that other purification tags, such as a his tag, can be used at the CBD position to mediate affinity purification.

In certain embodiments, the target protein contains the tripeptide CFN at the N-terminus after purification (Figure 3). The AS present in constructs 6-10 corresponds to the NheI site and was introduced to facilitate cloning. Cys + 1 is important for inactivating the intein under non-reducing conditions, presumably by a disulfide bond with Cys1. Although the functions of Phe + 2 and Asn + 3 are not known, these residues probably do not play an important role in Npu ^* C-terminal truncation activity, as previously reported for trans-splicing activity in wild-type NpuDnaE. The C-terminal cleavage efficiency depends not only on the immediate extein residue but also on the target protein (FIG. 20). This variability is due to steric hindrance by various target proteins in the association of C ^* and N, which can affect both the affinity of these two fragments as well as the intein catalytic efficiency.

  In certain embodiments, we have engineered the NpuDnaE intein with a rational design. This intein exhibits a rapid C-terminal cleavage reaction rate independent of N-terminal cleavage. We have shown the application of this engineered intein in protein purification. Comparing purification methods based on mutant NpuDnaE inteins with other purification methods using artificial split DnaB inteins, the method disclosed in the present invention eliminates the dependence of small peptides and provides even faster cleavage rates To do. Thus, the method disclosed in the present invention is useful in large-scale protein purification applications.

Example of purification by SIRP (FIG. 14): A soluble lysate containing NC1A-CBD dissolved in buffer A (NaCl 0.5M, Tris-HCl 10 mM, pH 8.0) was added to a disposable column containing 150 μL of chitin resin. And washed 4 times with 10 column volumes (CV) of buffer B (NaCl 0.5 M, NaPOi 50 mM, pH 6.0). All addition and washing steps were performed in the batch phase. The last wash was supplemented with ZnCl ₂ 0.5mM. Just prior to adding the lysate to the same chitin resin, the same concentration of ZnCl ₂ was added to the soluble lysate of C ^* -PTDH / GFP dissolved in buffer B. The column was then washed with 10 CV Buffer B containing ZnCl ₂ (0.5 mM) and finally incubated with 4 CV Buffer A containing 50 mM DTT for 30 minutes at room temperature or 3 hours at 6 ° C. Purified PTDH and GFP were collected in the flow-through. Traces of NC1A-CBD in the flow-through can be removed by passing through a fresh chitin column. In order to regenerate the NC1A-CBD-chitin affinity matrix, the resin used is thoroughly washed with buffer C (NaCl 1.5M, Na ₂ HPO ₄ 50 mM / NaOH, pH 11.4, ZnCl ₂ 0.5 mM). Bound C ^* was released. The regenerated column can be stored in storage buffer (NaCl 0.5M, Tris-HCl 10 mM, EDTA 1 mM, 0.15% NaN3, pH 8.0) at 4 ° C. for about 1 week without losing much activity. it can.

  Chemicals and strains: All chemicals were reagent grade and were purchased from Sigma-Aldrich (St. Louis, MO) or VWR International (Radnor, PA) unless otherwise noted. E. coli DH5α (Invitrogen, Grand Island, NY) was used for recombinant DNA cloning and manipulation. E. coli BLR (DE3) (Novagen, Madison, WI) was used for expression of the recombinant protein. ONPG was purchased from Research Products International Corp. (Mount Prospect, IL). Chitin beads were purchased from New England Biolabs (Ipswich, Mass.).

  Plasmid construction: A summary of the amino acid sequence, structure and numbering of a particular embodiment is shown in FIG.

To generate C-GFP (construct 1), the NpuC gene was amplified from plasmid KanR-IntRBS-NpuNC-CFN9 using primers NpuC_F_NdeI and OXP-NC-G-Rev, and GFP pet26-GFP by overlap extension PCR Was ligated to the N terminus of the plasmid using primers OXP-GFP-NC-FWD and XhoI_GFP_R, and cloned between the NdeI and XhoI sites of pET-26b (+) (Novagen, Madison, WI). Mutation D118G was introduced using site-directed mutagenesis to generate C ^* -GFP (3) with primers NpuCD17G-F and NpuCD17G-R.

  To make CBD-N (2), NpuN is also amplified from plasmid KanR-IntRBS-NpuNC-CFN9 using primers HindIII-Link-Npu F and NpuN_R_XhoI and binds to the chitin binding domain (CBD). Amplified from pTWIN1 (New England BioLabs) using primers NdeI-CBD-F and HindIII-CBD-R by overlap extension PCR and NdeI and XhoI sites of pET-26b (+) (Novagen, Madison, WI) Inserted between. CBD-NpuNC1A (4) was generated by site-directed mutagenesis using primers NheI-C1A-F and NpuN_R_XhoI.

  ELP-N (5) was constructed by inserting NpuN (amino acids 1-102) between the EcoRI and HindIII sites of plasmid pET-EI: MBP10. NpuN is first amplified using the primers HindIII-Link-Npu F and HindIII-6H-NupN-R, then the primers EcorI-Linker-NpuN F and HindIII-6H- to include restriction sites and a flexible linker. Amplification was performed again using NupN-R.

C ^* -DsRed (7) was cloned between the NdeI and XhoI sites of pET-26b (+) (Novagen, Madison, WI). NpuC ^* was amplified from C ^* -GFP using primers NpuC_F_NdeI and NheI-NpuC CFN-R. DsRed was amplified from pTY24 plasmid (NCRR, YRS, Seattle, WA) using primers HindII-L-DsRed-fwd and XhoI_DsRed_R. The product was coupled to NpuC ^* by digestion of the NheI enzyme, resulting in the short linker peptide CFNAS. Apart from the standard CFN sequence, an “AS” dipeptide corresponds to the NheI restriction site and was included to facilitate subsequent cloning.

To clone C ^* -PTDH (6), the phosphate dehydrogenase “PTDH” was amplified from plasmid PTDH12xA176R-pet15b11 using primers NheI-PTDH-F and XhoI-PTDH12x-R and digested with NheI and XhoI Inserted into NpuC ^* -DsRed (7). Plasmid construct ^{C * -β_Gal (8), C} * -CAT (9) and ^C * -MBP (10) was synthesized by inserting between the NheI and XhoI sites using appropriate primers in the same way. The β-galactosidase gene was amplified from the plasmid pET-EI: β-galactosidase. Similarly, chloramphenicol acetyltransferase (CAT) and maltose binding protein (MBP) genes were amplified from plasmids pET-EI: CAT and pET-E-MBP (a gift from Professor David Wood), respectively.

  Protein expression and purification: E. coli BLR (DE3) was transformed with the appropriate expression plasmid, tetracycline 5 μg / mL and ampicillin 100 μg / mL (FIG. 3, construct 5) or tetracycline 5 μg / mL and kanamycin 50 μg / mL (others) Agar plates containing the entire construct). The next day, one colony was picked and grown in 5 mL Luria-Bertani (LB) broth to an OD600 of about 0.6. The culture was transferred to 1 L LB broth containing the same antibiotic and grown at 37 ° C. until the OD600 was approximately 0.6. Protein expression was induced at 18 ° C. for 14 hours by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.2 mM). After expression, cells were harvested by centrifugation at 6000 × g for 15 minutes at 4 ° C. and stored at −80 ° C. until use.

  To purify CBD-N / NC1A (FIG. 3, constructs 2, 4), the cell pellet was reconstituted in buffer A (NaCl 0.5M, Tris-HCl 10 mM, pH 8.0) at 10 mL per gram wet pellet. Suspended and disrupted by sonication (QSonica Misonix 200, Amp 10, 16-20W, 1 second pulse 6 seconds rest for 1 minute). The soluble lysate was collected after centrifugation at 16,000 × g for 20 minutes at 4 ° C., passed through a 5-ml Ni-NTA column (GE Healthcare Life Sciences, Piscataway, NJ) and washed with a buffer of NaCl (NaCl 0). Washed with 5 M, Tris-HCl 10 mM, imidazole 45 mM, pH 8) and eluted with buffer A containing 150 mM imidazole.

Protein C / C ^* -GFP (FIG. 3, constructs 1, 3) is a similar method but buffer B (NaCl 0.5M, NaPOi 50 mM, pH 6.0, to minimize proteolysis). Purified using 1 × protease inhibitor or cocktail (Roche Applied Science, Indianapolis, Ind.). The purified protein was buffer exchanged into buffer A and concentrated by a 10 kDa ultrafiltration spin column (Amicon Ultra, Millipore, Billerica, Mass.).

  In the purification example using Method 1 (FIG. 16), the whole cell pellet was resuspended in Buffer A. In a purification example using Method 2, the whole cell pellet was resuspended in column buffer (NaCl 1M, Tris-HCl 10 mM, pH 8) to increase binding of the target protein to the chitin resin.

Intein kinetic characteristics: All experiments characterizing intein were performed using the purified protein diluted in buffer A at the indicated temperature with the indicated amount of reducing agent. All reactions contained 20 μM of each intein fragment. Samples were taken at various times after the start of the reaction and unless otherwise stated 2 × SDS sample buffer (Tris-HCl 0.5M, pH 6.8, 20% glycerol, 10% w / v SDS, 0.1% w / V bromophenol blue, 2% β-mercaptoethanol), incubated at 95 ° C. for 5 minutes and analyzed using a 12% SDS-PAGE gel. The gel was stained with Coomassie Brilliant Blue R250. For samples corresponding to the 0 minute time point, purified C / C ^* -GFP (constructs 1 and 3) protein is first mixed with 2 × SDS sample in the absence of β-mercaptoethanol and incubated at 95 ° C. for 3 minutes. did. CBD-N / NC1A (FIG. 3, constructs 2 and 4) and β-mercaptoethanol were then added to the mixture. The entire mixture was incubated at 95 ° C. for an additional 3 minutes to inactivate the protein. Band intensities corresponding to reactants and products were quantified using the Trace Quantity module in Quantity One software (BioRad, Hercules, CA).

Protein purification by reversible precipitation of elastin-like peptides: In this embodiment, ELP-N (FIG. 3, construct 5) was pre-purified by one ammonium sulfate precipitation to facilitate the decoding of SDS-PAGE gels. It has been found that the efficiency of protein purification is not improved. Clarified cell lysates of ELP-N and C ^* -POI (FIG. 3, constructs 3, 6-10) were mixed thoroughly and incubated at room temperature for 10 minutes to allow C ^* and N to associate (FIG. 17A, lane 3). Ammonium sulfate (final concentration 0.4M) was added to the mixture to induce precipitation of the ELP complex. The pellet containing the target protein non-covalently bound to ELP was resuspended in buffer A (FIG. 17A, lane 5). The intein reaction was started by adding DTT (50 mM) and carried out at room temperature for 3 or 20 hours. Lower DTT concentrations can be used to induce C-terminal truncation (Figure 7). At the end of the reaction, ammonium sulfate (0.4M) was added to the mixture to precipitate the ELP-N / C ^* complex. This sediment was removed by centrifugation. The supernatant contained highly purified target protein (Figure 17A, lanes 8, 9).

Protein purification with chitin resin: The slurry of chitin beads is first incubated with CBD-N lysate (Figure 3, Construct 2) for 10 minutes at room temperature and concentrated with column buffer to remove all contaminating proteins. Washed (FIG. 17C, lane 3), then lysate containing C ^* -GFP was added (FIG. 3, construct 3). After washing, DTT (5 mM) was added to the mixture to induce C-terminal cleavage. The reaction was essentially complete after 3 hours of reaction at room temperature (FIG. 17C, lane 5) and purified GFP was collected in the flow-through (FIG. 17C, lane 7). CBD-N and a small amount of unreacted C ^* -GFP were still bound to the chitin beads (FIG. 17C, lane 6). The cleaved C ^* is so small in size (4 kDa) that it cannot be visualized on the gel, but it was estimated that it would remain on the column due to interaction with N.

  Molecular modeling: The structures of mini-MtuRecA (pdb: 2IMZ), NpuDnaE (pdb: 2KEQ) and SspDnaE (pdb: 1ZD7) are visualized using Visual Molecular Dynamics (VMD) and aligned using the MultiSeq module in VMD I let you. Hydrogen bond interactions were identified by VMD. The NMR structure of NpuDnaE includes 20 different solution structures. For clarity, only the alignment of structure # 7 of SspDnaE and NpuDnaE is shown in FIG. 5C.

Temperature-dependent kinetics: using Lab Fit software package (Campina Grande, Paraiba, Brazil) to determine the half-life of C ^* -GFP C-terminal cleavage reaction at different temperatures, all data in FIG. 7C A trend line that best matches the point was created. The time corresponding to 50% cut was estimated based on the approximate curve.

Estimated half-life of C ^* -GFP cleavage at various temperatures:
Temperature Half-life 4 ° C 243 minutes 16 ° C 70 minutes 22 ° C 55 minutes 37 ° C 16 minutes

  Quantification of purified protein content: Target protein purification yield was quantified by measuring the concentration of the purified sample using the Bradford method (Coomassie Plus Bradford Assay Reagent, Pierce Biotechnology, Rockford, IL). To estimate percent recovery, soluble lysate and purified protein were added to the same SDS-PAGE, stained with SimplyBlue SafeStain (Life Technology, Carlsbad, Calif.), And the band intensity corresponding to the target protein was determined by Quantity One software. (BioRad, Hercules, Calif.) Using the Trace Quantity module.

  Prepurification of ELP-N: Ammonium sulfate (0.4M) was added to the soluble lysate to induce ELP-N phase separation. The mixture was incubated at room temperature for about 3 minutes and centrifuged at 14,000 × g for 10 minutes. The resulting pellet was resuspended in 1/3 of the original volume of buffer A. A low intensity water bath sonicator (Ultrasonic Cleaner, GB928) was used (5 minutes) to assist in the resuspension of ELP-N.

  Measurement of sample protein activity: The activity of purified PTDH was confirmed by NBT assay as described by Mayer et al. Since DTT interferes with the NBT reaction at high concentrations, the DTT concentration in the purified protein is buffered using a 30 kDa cut-off spin column (Amicon Ultra-15 Centrifugal Filter Unit, Millipore, Billerica, Mass.) Prior to measurement. Reduced to about 5 μM by exchange.

  The activity of MBP was confirmed by binding to amylose resin (New England Biolabs, Ipswich, Mass.). Amylose beads (25 μL) were incubated with purified protein (500 μL) for 10 minutes at room temperature, washed twice with 500 μL of buffer A and resuspended in 500 μL of buffer A. 10 μL of this suspension was mixed with 10 μL of 2 × SDS addition buffer, boiled at 95 ° C. for 3 minutes, and analyzed by SDS-PAGE. MBP protein could be visualized in amylose beads suspension but not in wash buffer.

  Proteins GFP and DsRed were assayed by fluorescence measurements against the non-fluorescent protein CAT. Purified GFP or DsRed was diluted 2-fold and transferred to a 96-well plate (150 μL / well). Fluorescence intensity was measured using a fluorimeter SpectraMax Gemini EM (Molecular Devices, Sunnyvale, Calif.) At excitation / emission wavelengths of 485/538 nm (GFP) or 544/590 nm (DsRed). The control protein CAT produced background values in both assays.

  β-galactosidase activity was measured by hydrolyzing o-nitrophenyl β-D-galactopyranoside (ONPG) to o-nitrophenol having an absorption at 420 nm. The purified β-galactosidase was diluted 1000 times with Z buffer (Na2HPO4 0.06M, NaH2PO4 0.04M, KCl 0.01M, MgSO4 0.001M and 0.27% 2-mercaptoethanol). Diluted protein (30 μL) was mixed with Z buffer (200 μL) and ONPG (70 μL, 4 mg per mL of potassium phosphate buffer 100 mM pH 7) and incubated at 22 ° C. for 15 or 30 minutes. At the end of the reaction, 500 μL of stop buffer (Na 2 CO 3 1M) was added and the absorbance at 420 nm was measured with a Biomate 3 spectrophotometer (Thermo Electron Corporation).

  To estimate the enzyme unit of β-galactosidase, the following equation was used:

8 × 10 ⁵ nanoliters is the volume of the reaction; 4500 M−1 cm−1 is the extinction coefficient of o-nitrophenol and 1-cm is the length of the optical path. One unit of β-galactosidase is defined as the amount of enzyme required to hydrolyze 1 micromole of ONPG per minute at 22 ° C.

  To estimate the sample recovery of β-galactosidase, a similar activity assay was performed in 96-well plates by diluting purified β-galactosidase 1000-fold with Z buffer. Diluted protein (50 μL) was mixed with Z buffer (50 μL) and 10 μl of ONPG solution. Absorption at 420 nm was measured after incubation for 20 minutes using a SpectraMax 340PC384 absorbance microplate reader (Molecular Devices, Sunnyvale, Calif.).

  Any embodiment described herein can be practiced with respect to any method, kit, reagent or composition of the invention, and vice versa. Furthermore, the compositions of the present invention can be used to implement the methods of the present invention.

  It will be understood that the particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be used in various embodiments without departing from the scope of the invention. One skilled in the art will recognize that there are numerous equivalents to the specific methods described herein, or will be able to confirm using only routine experimentation. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

  All publications and patent applications mentioned in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

  When used in conjunction with the term “comprising” in the claims and / or the specification, the use of the word “a” or “an” means “one”. ", But also matches the meaning of" one or more "," at least one "and" one or more than one " To do. The term “or” in the claims means that only the option or the options are mutually exclusive, even though the disclosure supports a definition that only means the option and “and / or”. Is used to mean "and / or" unless explicitly indicated. Throughout this specification, the term “about” is used to indicate that a value includes the variation in error inherent in the device, method, or study object used to measure the value. Used.

  As used herein and in the claims, “comprising” (and any form of “comprising” including “comprise” and “comprises”), “having ( having "(and any form of having) and" including "(and" includes "and" include "). "Including any form") or "containing" (and any form containing "contains", "contain", etc.) The terms are inclusive or non-limiting and do not exclude additional elements or method steps not listed. As used herein, the phrase “consisting essentially of” refers to a specified material or step and that does not substantially affect the basic and novel characteristics of the claimed invention. Limit the scope of the claims. As used herein, the expression “consisting of” excludes any element, step, or ingredient not specified in the claim, for example, an impurity usually associated with the element or limitation. To do.

  As used herein, the term “or combinations thereof” means all permutations and combinations of the items listed before the term. For example, “A, B, C, or a combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context , BA, CA, CB, CBA, BCA, ACB, BAC or CAB. This example is also explicitly followed by combinations containing one or more repetitions of one or more items or terms such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB. Those skilled in the art will appreciate that there is usually no limit on the number of items or terms in any combination, unless otherwise apparent from the context. In certain embodiments, the present invention can also include methods and compositions that can also use the transitional phrase “consisting essentially of” or “consisting of”.

  As used herein, terms indicating approximations such as, but not limited to, “about”, “substantial”, or “substantially” are intended to It does not need to be absolute or complete, but means a condition when it is nearly sufficient to consider it reasonable to specify a condition as suggested by one skilled in the art. The variable range of the description will depend on how large the change can occur, and those skilled in the art will still understand that the modified features still have the necessary properties and capabilities of the unmodified features. It is the range that can be recognized. In general, although not yet discussed, the numerical values herein modified by approximate terms such as “about” are at least ± 1, 2, 3, 4, 5 from the values presented. , 6, 7, 10, 12 or 15%.

  All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation with respect to the present disclosure. While the compositions and methods of the present invention have been described using preferred embodiments, those skilled in the art will understand the compositions and / or described herein without departing from the concept, spirit and scope of the present invention. It will be understood that changes may be made in the method and sequence of steps or method steps. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Table of sequences used Construct 1
C-GFP:
(SEQ ID NO: 1)
Translated C-GFP:
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCFNVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELLEHHHHHH (SEQ ID NO: 2)
Structure 2
CBD-N
(SEQ ID NO: 3)
Translated CBD-N:
MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQEACGGGGSGGGGSASCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQHLPIDEIFERELH
Construction 3
C ^* -GFP:
(SEQ ID NO: 5)
Translated C ^* -GFP:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNCFNVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELLEHHHHHH (SEQ ID NO: 6)
Structure 4
CBD-NC1A
(SEQ ID NO: 7)
Translated CBD-NC1A:
MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQEACGGGGSGGGGSASALSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQHLPHIDHELH
Construction 5
ELP-N
(SEQ ID NO: 9)
Translated ELP-N:
(SEQ ID NO: 10)
Construction 6
C ^* -PTDH:
(SEQ ID NO: 11)
Translated C ^* -PTDH:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNCFNASMLPKLVITHRVHEEILQLLAPHCELITNQTDSTLTREEILRRCRDAQAMMAFMPDRVDADFLQACPELRVIGCALKGFDNFDVDACTARGVWLTFVPDLLTVPTAELAIGLAVGLGRHLRAADAFVRSGKFRGWQPRFYGTGLDNATVGFLGMGAIGLAMADRLQGWGATLQYHARKALDTQTEQRLGLRQVACSELFASSDFILLALPLNADTLHLVNAELLALVRPGALLVNPCRGSVVDEAAVLAALERGQLGGYAADVFEMEDWARADRPQQIDPALLAHPNTLFTPHIGSAVRAVRLEIERCAAQNILQALAGERPINAVNRLPKANPAADLEHHHHHH (SEQ ID NO: 12)
Construction 7
C ^* -DsRed
(SEQ ID NO: 13)
Translated C ^* -DsRed:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNCFNASASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHHLFLLEHHHHHH (SEQ ID NO: 14)
Construction 8
C ^* -β-Gal
(SEQ ID NO: 15)
Translated C ^* -β-Gal:
(SEQ ID NO: 16)
Construction 9
C ^* -CAT
(SEQ ID NO: 17)
Translated C ^* -CAT:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNCFNASMEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFLKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGALEHHHHHH (SEQ ID NO: 18)
Construct 10
C ^* -MBP
(SEQ ID NO: 19)
Translated C ^* -MBP:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNCFNASMKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRGLEHHHHHH (SEQ ID NO: 20)
Construct 11
N-CBD:
(SEQ ID NO: 21)
Translated N-CBD:
MCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNKLGGGGSGGGGSASMKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSH
Construct 12
NC1A-CBD:
(SEQ ID NO: 23)
Translated NC1A-CBD:
MALSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNKLGGGGSGGGGSASMKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSH
Construction 13
C-PTDH:
(SEQ ID NO: 25)
Translated C-PTDH:
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCFNASMLPKLVITHRVHEEILQLLAPHCELITNQTDSTLTREEILRRCRDAQAMMAFMPDRVDADFLQACPELRVIGCALKGFDNFDVDACTARGVWLTFVPDLLTVPTAELAIGLAVGLGRHLRAADAFVRSGKFRGWQPRFYGTGLDNATVGFLGMGAIGLAMADRLQGWGATLQYHARKALDTQTEQRLGLRQVACSELFASSDFILLALPLNADTLHLVNAELLALVRPGALLVNPCRGSVVDEAAVLAALERGQLGGYAADVFEMEDWARADRPQQIDPALLAHPNTLFTPHIGSAVRAVRLEIERCAAQNILQALAGERPINAVNRLPKANPAADLEHHHHHH (SEQ ID NO: 26)
Construct 14
C ^* -A-GFP:
(SEQ ID NO: 27)
Translated C ^* -A-GFP:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNAFNVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELLEHHHHHH (SEQ ID NO: 28)
Construct 15
C ^* -D-GFP:
(SEQ ID NO: 29)
Translated C ^* -D-GFP:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNDFNVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELLEHHH
HHH (SEQ ID NO: 30)
Construct 16
C ^* -L-GFP:
(SEQ ID NO: 31)
Translated C ^* -L-GFP:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNLFNVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELLEHHHHHH (SEQ ID NO: 32)
Construct 17
C ^* -P-GFP:
(SEQ ID NO: 33)
Translated C ^* -P-GFP:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNPFNVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELLEHHHHHH (SEQ ID NO: 34)
Construct 18
C ^* -R-GFP:
(SEQ ID NO: 35)
Translated C ^* -R-GFP:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASNRFNVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELLEHHHHHH (SEQ ID NO: 36)
Translated C ^* -:
MIKIATRKYLGKQNVYGIGVERDHNFALKNGFIASN (SEQ ID NO: 37)
The amino acid sequence of ELP:
(SEQ ID NO: 38)
Amino acid sequence of intein N fragment:
ALSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN (SEQ ID NO: 39)
C ^* -DNA:
ATGATCAAAATAGCCACACGTAAATATTTAGGCAAACAAAATGTCTATGGCATTGGAGTTGAGCGCGACCATAATTTTGCACTCAAAAATGGCTTCATAGCTTCTAAT
(SEQ ID NO: 40)
ELP-intein N fragment:
(SEQ ID NO: 41).
Translated ELP-intein N fragment:
(SEQ ID NO: 42)
Primers used in this study:
NPUC_F_NDEI 104:
TTAGAAGGCATATGATCAAAATAGCCACACGTAAATATTTAGG (SEQ ID NO: 43).
OXP-NC-G-REV:
CCTCGCCCTTGCTCACATTGAAACAATTAGAAGCTATGAAGCCAT (SEQ ID NO: 44).
OXP-GFP-NC-FWD:
ATAGCTTCTAATTGTTTCAATGTGAGCAAGGGCGAGG (SEQ ID NO: 45).
XHOI_GFP_R:
TAAAATCTCGAGTAACTCGTCCATGCCGAGAG (SEQ ID NO: 46).
NPUCD17G-F:
GGCAAACAAAATGTCTATGGCATTGGAGTT (SEQ ID NO: 47).
NPUCD17G-R:
GTCGCGCTCAACTCCAATGCCATAGACATT (SEQ ID NO: 48).
HINDIII-LINK-NPU F:
CCTGGAAGCTTGTGGAGGCGGAGGGAGCGGAGGCGGAGGGAGCGCTAGCTGTTTAAGCTATGAAACGGAAATATTGAC (SEQ ID NO: 49).
NPUN_R_XHOI:
ATATAGCTCGAGATTCGGCAAATTATCAACCCG (SEQ ID NO: 50).
NDEI-CBD-F:
TAATTTAACATATGAAAATCGAAGAAGGTAAACTGACAAATCCT (SEQ ID NO: 51).
HINDIII-CBD-R:
AAGATTAAAGCTTCTTGAAGCTGCCACAAGGCA (SEQ ID NO: 52).
NHEI-C1A-F:
AATTAAGCTAGCGCCTTAAGCTATGAAACGGAAATATTGACA (SEQ ID NO: 53).
ECORI-LINKER-NPUN F:
AATATGGGAATTCGGAGGCGGAGGGAGCGG (SEQ ID NO: 54).
HINDIII-6H-NUPN- R:
GTACATTAAGCTTAGCAGCCGGATCTCAGT (SEQ ID NO: 55).
NHEI-NPUC CFN-R:
ATTCGCGCTAGCATTGAAACAATTAGAAGCTATGAAGCC (SEQ ID NO: 56).
XHOI_DSRED_R:
TAAAATCTCGAGCAGGAACAGGTGGTGGC (SEQ ID NO: 57).
HINDII-L-DSRED-FWD:
TTCAATAAGCTTGGAGGCGGAGGGAGCGGAGGCGGAGGGAGCGCTAGCGCCTCCTCCGAGGACG (SEQ ID NO: 58).
NHEI-PTDH-F:
ATTTAACGCTAGCATGCTGCCGAAACTCGTTATAACTC (SEQ ID NO: 59).
XHOI-PTDH12X-R:
AGTTTAGCTCGAGGTCTGCGGCAGGATTGG (SEQ ID NO: 60).
NHEI-LACZ-F:
ATTTCAATGCTAGCATGACCATGATTACGGATTCACT (SEQ ID NO: 61).
XHOI-LACZ-R:
TGATAATCTCGAGTTTTTGACACCAGACCAACTG (SEQ ID NO: 62).
NHEI-CAT-F:
GTTTCAATGCTAGCATGGAGAAAAAAATCACTGGATATACCACCGTTGATATAT (SEQ ID NO: 63).
XHOI-CAT-R:
TAATAATTAACTCGAGCGCCCCGCCCTGCCAC (SEQ ID NO: 64).
NHEI-MBP-F:
GTTTCAATGCTAGCATGAAAATCGAAGAAGGTAAACTGGTAATCT (SEQ ID NO: 65).
XHOI-MBP-R:
AAGTTATACTCGAGTCCCCTTCCCTCGATCC (SEQ ID NO: 66).

Claims (58)

A method for purifying a desired protein (POI) comprising:
A first fusion protein containing POI fused to the C-terminus of an intein C fragment is contacted with a second fusion protein containing an intein N fragment and a purification tag, and the first fusion protein and the second Forming a complex with the fusion protein;
Cleaving the POI from the intein C fragment, releasing the protein from the complex;
Isolating said POI.   The method of claim 1, wherein the intein is a split intein.   The method according to claim 1, wherein the intein is Nostoc punctiforme naturally split intein DnaE.   Intein is Sine of Synechocystis sp., Aha of Aphanothiki halophytica, Aov of Aphanizomenon Ovalisporum, Asp of Anabaena spp. (CCYOOlO), Csp of Cyanoseis sp. (PCC8801), Cwa of Crocosphaela watsonii, Maer of Microcystis aeruginosa (NIES843), Mcht (PCC7420) -2 of Microcholes xonoplastes, Oli of Oclatria Rimnetica, Synecococcus Elongatas Sel (PC7942), Synechococcus sp. Ssp (PCC7002), Thermocinecococcus elongatas Tel of Thernlosynechococcus elongates), it is selected from the group consisting of Tvu of Ter-3 of Torikodesumiumu-Erisuraeumu, and thermo Synechococcus, Vulcan (Thernlosynechococcus vulcanus), The method of claim 1.   The method of claim 1, wherein the intein C fragment has a mutation that significantly delays N-terminal cleavage, suppresses trans-splicing ability, and increases C-terminal cleavage rate and efficiency compared to an unmutated intein C fragment. .   The method according to claim 1, wherein the intein is a naturally split intein DnaE, and the C-intein fragment has an Asp118Gly mutation in the C-intein fragment.   The method of claim 1, wherein the intein C fragment comprises the amino acid sequence of SEQ ID NO: 37.   2. The method of claim 1, wherein the purification tag is located at the intein split junction at the C-terminus of the intein N fragment.   The method according to claim 1, wherein the intein N fragment has a mutation that eliminates N-terminal cleavage activity.   The method of claim 1, wherein the intein N fragment comprises the amino acid sequence of SEQ ID NO: 39.   2. The purification tag is an affinity tag selected from the group consisting of a chitin binding domain (CBD), 6x histidine, maltose binding domain (MBP), glutathione-S-transferase (GST), and combinations thereof. The method described in 1.   2. The method of claim 1, wherein the purification tag is an affinity tag selected from the group consisting of SEQ ID NO: 38.   2. The method of claim 1, wherein the second fusion protein comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 10, 24, and combinations thereof.   The method of claim 1, wherein the purification tag is an elastin-like peptide (ELP).   The method of claim 1, wherein the purification tag is a precipitation tag comprising the amino acid sequence of SEQ ID NO: 38. The purification tag is a sedimentation tag, and the method precipitates the complex;
Washing the complex;
Solubilizing the complex;
The method of claim 1, further comprising inducing intein cleavage.   17. A method according to claim 16, wherein sedimentation of the complex or washing of the complex comprises contacting the complex with one or more cleavage inhibitors. The purification tag is an affinity tag, and the method binds the complex to an affinity resin capable of binding to the affinity tag;
Washing the complex with a washing buffer before the cutting step;
The method of claim 1, further comprising inducing intein cleavage.   19. The method of claim 18, wherein binding of the complex or washing of the complex comprises contacting the complex with one or more cleavage inhibitors.   19. The method of claim 18, wherein inducing intein cleavage comprises contacting the complex with one or more reducing or chelating agents.   Induction of intein cleavage is selected from the group consisting of a complex and ethylene glycol aminoethyl ester tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), dipicolinic acid (DPA), nitrilotriacetic acid (NTA) 1 or 2 19. A method according to claim 18, comprising contacting the chelating agent. Further comprising incubating the complex with a first wash buffer prior to inducing cleavage, wherein the wash buffer inhibits cleavage and / or Zn ²⁺ , Cu ² , Mg ²⁺ , Co ²⁺ , Mn ²⁺ And a cleavage inhibitor selected from the group consisting of Fe ²⁺ .   The method of claim 1, further comprising washing the complex with a first wash buffer prior to inducing cleavage, wherein the wash buffer comprises a cleavage inhibitor that inhibits a C-terminal cleavage reaction.   The method of claim 1, wherein the induction of C-terminal protein cleavage comprises induction of sulfur-induced C-terminal cleavage. Induction of C-terminal protein cleavage is selected from the group consisting of DTT, Zn ²⁺ chelator, trialkylphosphine (tris (2-carboxyethyl) phosphine (TCEP), 2-mercaptoethanol, cysteine and combinations thereof. 2. The method of claim 1, comprising induction of sulfur-induced C-terminal truncation in the presence of an agent.   2. The method of claim 1, wherein the induction of C-terminal protein cleavage comprises induction of intein cleavage by chelating a cleavage inhibitor using a chelating agent. The purification tag is an affinity tag, and the method further comprises the step of binding the complex to an affinity resin, wherein the separation of the POI from the complex comprises the POI from the affinity resin to which the complex is bound. The method of claim 1, comprising separating. The purification tag is a sedimentation tag, and the method is a step of sedimenting the complex to form a sedimented complex,
Separating the POI from the complex comprises solubilizing the precipitated complex to form a solubilized complex;
2. The method of claim 1, further comprising separating the POI from the solubilized complex.   The method of claim 1, further comprising regenerating the second fusion protein by dissociating an intein C fragment from the second fusion protein.   POI is a bioactive peptide, enzyme, enzyme inhibitor, enzyme catalytic site, DNA binding protein, isolated protein domain, receptor ligand, receptor, growth factor, cytokine, structural protein, antibody, antibody fragment, epitope, 2. The method of claim 1, wherein the method is selected from epitope-binding regions, antigens, allergens and consecutive or overlapping fragments of the desired protein sequence. Binding the complex to an affinity resin before the purification tag is an affinity tag and the method induces C-terminal protein cleavage;
The method of claim 1, further comprising regenerating the affinity resin by dissociating an intein C fragment from a second fusion protein.   Regenerating the second fusion protein by dissociating an intein C fragment from the second fusion protein; and re-contacting the regenerated second fusion protein with the first fusion protein. The method of claim 1.   The purification tag is an affinity tag, and the second fusion protein is bound to an affinity resin selected from the group consisting of chitin beads, nickel resin, amylose resin, glutathione, and combinations thereof. The method described.   2. The method of claim 1, wherein the purification tag is a precipitation tag that mediates precipitation of the second fusion protein and the complex is precipitated. A method for purifying a desired protein (POI) comprising:
Providing a first fusion protein comprising the POI and an intein C fragment, wherein the POI is fused to the C-terminus of the intein C fragment, and the intein is a naturally split intein DnaE, the intein C A fragment has an Asp118Gly mutation in said intein C fragment;
Providing a second fusion protein comprising an intein N fragment and a purification tag, wherein the purification tag is inserted into an intein split junction at the C-terminus of the intein N fragment, wherein the intein N fragment is N-terminally cleaved Having a mutation that abolishes activity;
Contacting the first fusion protein with the second fusion protein in a binding buffer, wherein the second fusion protein is attached to a resin that binds to the purification tag; Can bind specifically to a purified resin, a complex of the first fusion protein and the second fusion protein is formed, and the binding buffer is between the POI and the intein C fragment. Inhibiting the C-terminal protein cleavage of the first fusion protein of
Inducing C-terminal protein cleavage of the first fusion protein between the POI and the intein C fragment, thereby releasing the POI;
Separating the POI from the C-terminus of the first fusion protein and the intein C fragment. A method for purifying a desired protein (POI) comprising:
Providing a first fusion protein comprising a POI and an intein C fragment, wherein the POI is fused to the C-terminus of the intein C fragment and the intein is a naturally split intein DnaE, wherein the intein C fragment is Having an Asp118Gly mutation in the intein C fragment;
Providing a second fusion protein comprising an intein N fragment and a precipitation tag, wherein the precipitation tag is inserted into an intein split junction, which is the C-terminus of the intein N fragment, and the intein N fragment has N-terminal cleavage activity Having a mutation that eliminates
Contacting the first fusion protein and the second fusion protein in a binding buffer, wherein a complex of the first fusion protein and the second fusion protein is formed; The binding buffer inhibiting C-terminal protein cleavage of the first fusion protein between the POI and the intein C fragment;
Precipitating a complex of the first fusion protein and the second fusion protein;
Solubilizing the complex in a low salt buffer;
Inducing C-terminal protein cleavage of the first fusion protein between the POI and the intein C fragment, thereby releasing the POI;
Separating the POI from the complex of the first fusion protein and the second fusion protein by a second sedimentation.   A fusion protein comprising a desired protein (POI) and an intein C fragment, wherein the POI is fused to the C-terminus of the intein C fragment, and the intein is a naturally split intein DnaE, and the intein C fragment Said fusion protein having an Asp118Gly mutation in said intein C fragment.   38. The fusion protein of claim 37, comprising SEQ ID NO: 39.   POI is bioactive peptide, enzyme, enzyme inhibitor, enzyme catalytic site, DNA binding protein, isolated protein domain, receptor ligand, receptor, growth factor, cytokine, antibody, antibody fragment, epitope, epitope binding region 38. The fusion protein of claim 37, selected from consecutive or overlapping fragments of the antigen, allergen and desired protein sequence.   A fusion protein comprising an intein N fragment and a purification tag, wherein the purification tag is located at an intein split junction that is the C-terminus of the intein N fragment, and the intein N fragment loses N-terminal cleavage activity The fusion protein comprising:   41. The fusion protein of claim 40, comprising SEQ ID NO: 39.   41. The fusion protein of claim 40, comprising SEQ ID NO: 4, 10, 24 or a combination thereof. A vector comprising a first DNA element encoding the C-terminus of an intein C fragment operably linked to a promoter comprising:
The intein C fragment has a mutation that suppresses N-terminal cleavage and increases C-terminal cleavage compared to an unmutated intein C fragment, and a second DNA element encoding a desired protein (POI) The vector having a cloning site that allows insertion of the intein C fragment into the C-terminus.   44. The vector of claim 43, wherein the intein is Nostoc punctiforme naturally split intein DnaE, and the C-intein fragment has an Asp118Gly mutation in the C-intein fragment.   44. The vector of claim 43, wherein the first DNA element encodes the amino acid sequence of SEQ ID NO: 37.   44. The vector of claim 43, wherein the first DNA element comprises SEQ ID NO: 40.   POI is bioactive peptide, enzyme, enzyme inhibitor, enzyme catalytic site, DNA binding protein, isolated protein domain, receptor ligand, receptor, growth factor, cytokine, antibody, antibody fragment, epitope, epitope binding region 44. The vector of claim 43, wherein the vector is selected from consecutive or overlapping fragments of the antigen, allergen and desired protein sequence. A vector comprising a DNA element encoding a fusion protein comprising an intein N fragment operably linked to a promoter and a purification tag comprising:
The vector, wherein the purification tag is located at an intein split junction that is the C-terminus of the intein N fragment, and the intein N fragment has a mutation that eliminates N-terminal cleavage activity.   49. The vector of claim 48, wherein the purification tag is an affinity tag.   49. The vector of claim 48, wherein the purification tag is a precipitation tag.   49. The vector of claim 48, wherein the DNA element comprises SEQ ID NO: 23 or SEQ ID NO: 41. A kit for isolating a desired protein (POI) comprising:
A first vector comprising a first DNA element encoding the C-terminus of an intein C fragment operably linked to a promoter, wherein the intein C fragment is N-terminally cleaved compared to an unmutated intein C fragment The first vector has a cloning site that has a mutation that reduces C-terminal cleavage and increases C-terminal truncation, and wherein the first vector allows insertion of a second DNA element encoding POI into the C-terminus of the intein C fragment. 1 vector,
A second vector comprising a second DNA element encoding a fusion protein comprising an intein N fragment operably linked to a promoter and a purification tag, wherein the purification tag is at the C-terminus of the intein N fragment A second vector having a mutation located at the split junction, wherein the intein N fragment has a mutation that eliminates N-terminal cleavage activity; or an intein N fragment and a purification tag located at the intein split junction at the C-terminus of the intein N fragment Wherein the intein N fragment has a mutation that eliminates N-terminal cleavage activity; and
Instructions for inserting the DNA element encoding the POI into the cloning site of the first vector;
The kit comprising instructions for isolating the POI. A method for purifying a desired protein (POI) comprising:
A first fusion protein containing the POI fused to the C-terminus of an intein C fragment is contacted with a second fusion protein containing an intein N fragment and a purification tag, so that the first fusion protein and the second fusion A step of forming a complex with a protein, wherein the intein C fragment significantly delays N-terminal cleavage as compared to an intein C fragment that is not mutated, suppresses trans-splicing ability, and reduces C-terminal cleavage rate and efficiency. Having a mutation that increases; and
Cleaving the POI from the intein C fragment, releasing the protein from the complex;
Isolating said POI.   The method according to claim 1, wherein the intein is a naturally split intein DnaE, and the C-intein fragment has an Asp118Gly mutation in the C-intein fragment. A method for purifying a desired protein (POI) comprising:
Providing a first fusion protein comprising said POI and intein C fragment, wherein said POI is intein DnaE fused to the C-terminus of said intein C fragment and intein is split naturally, said intein C The fragment has an Asp118Gly mutation in said intein C fragment;
Providing a second fusion protein comprising an intein N fragment and a purification tag, wherein the intein N fragment has a mutation that eliminates N-terminal cleavage activity;
Contacting the first fusion protein and the second fusion protein in a binding buffer, wherein the second fusion protein is attached to a resin that binds to a purification tag, and the purification tag comprises: Can bind specifically to a purified resin, a complex of the first fusion protein and the second fusion protein is formed, and the binding buffer is between the POI and the intein C fragment. Inhibiting the C-terminal protein cleavage of the first fusion protein of
Inducing C-terminal protein cleavage of the first fusion protein between the POI and the intein C fragment, thereby releasing the POI;
Separating the POI from the C-terminus of the first fusion protein and the intein C fragment.   A fusion protein comprising a desired protein (POI) and an intein C fragment, wherein the POI is fused to the C terminus of the intein C fragment, and the intein is a naturally split intein DnaE, and the intein C fragment is A fusion protein having an Asp118Gly mutation in the intein C fragment. A kit for isolating a desired protein (POI) comprising:
A first vector comprising a first DNA element encoding the C-terminus of an intein C fragment operably linked to a promoter, wherein the intein C fragment has an Asp118Gly mutation in the intein C fragment; A first vector having a cloning site that allows insertion of a second DNA element encoding a POI into the C-terminus of said intein C fragment;
A second vector comprising a second DNA element encoding a fusion protein comprising an intein N fragment and a purification tag operably linked to a promoter; or a fusion protein comprising an intein N fragment and a purification tag;
Instructions for inserting the DNA element encoding the POI into the cloning site of the first vector;
The kit comprising instructions for isolating the POI. A method for purifying a desired protein (POI) comprising:
A first fusion protein containing the POI fused to the C-terminus of an intein C fragment is contacted with a second fusion protein containing an intein N fragment and a purification tag, and the first fusion protein and the second Forming a complex with the fusion protein of wherein the purification tag is located at the intein split junction which is the C-terminus of the intein N fragment;
Cleaving the POI from the intein C fragment, releasing the protein from the complex;
Isolating said POI.